Anti-glucosaminidase passive immunization for staphylococcus aureus infections

ABSTRACT

The present invention is directed to a monoclonal antibody that binds specifically to a  Staphylococcus aureus  glucosaminidase and inhibits in vivo growth of  S. aureus.  Also disclosed are monoclonal antibody binding portions, recombinant or hybridoma cell lines, pharmaceutical compositions containing the monoclonal antibody or binding portions thereof, and methods of treating 5*.  aureus  infection and osteomyelitis, and methods for introducing an orthopedic implant into a patient using the monoclonal antibody, binding portion, or pharmaceutical composition of the present invention.

This invention was made with government support under grant number R43AI085844 awarded by the National Institutes of Health. The governmenthas certain rights in this invention.

This application claims the benefit of U.S. Provisional PatentApplication Ser. No. 61/330,568, filed May 3, 2010, which is herebyincorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to passive immunization againstStaphylococcus aureus infection, particularly for the prevention ortreatment of osteomyelitis and for implantation of an orthopedic implantor graft. Antibodies that bind specifically to S. aureus glucosaminidaseand pharmaceutical compositions containing the same can be used forthese purposes.

BACKGROUND OF THE INVENTION

There is a great need for novel interventions of chronic osteomyelitis(OM) as approximately 112,000 orthopedic device-related infections occurper year in the US, at an approximate hospital cost of $15,000-70,000per incident (Darouiche, “Treatment of Infections Associated WithSurgical Implants,” N. Engl. J. Med. 350(14):1422-9 (2004)). Althoughimprovements in surgical technique and aggressive antibiotic prophylaxishave decreased the infection rate following orthopedic implant surgeryto 1-5%, osteomyelitis (OM) remains a serious problem and appears to beon the rise from minimally invasive surgery (Mahomed et al., “Rates andOutcomes of Primary and Revision Total Hip Replacement in the UnitedStates Medicare Population,” J. Bone Joint Surg. Am. 85(A-1):27-32(2003); WHO Global Strategy for Containment of Antimicrobial Resistance,2001). The significance of this resurgence, 80% of which is due toStaphylococcus aureus, is amplified by the fact that ˜50% of clinicalisolates are methicillin resistant S. aureus (MRSA). While the infectionrates for joint prosthesis and fracture-fixation devices have been only0.3-11% and 5-15% of cases, respectively, over the last decade (Lew andWaldvogel, “Osteomyelitis,” Lancet 364(9431):369-79 (2004); Toms et al.,“The Management of Peri-Prosthetic Infection in Total JointArthroplasty,” J. Bone Joint Surg. Br. 88(2):149-55 (2006)), this resultmay lead to amputation or death. Additionally, the popularization of“minimally invasive surgery” for elective total joint replacements (TJR)in which the very small incision often leads to complications from theprosthesis contacting skin during implantation, has markedly increasedthe incidence of OM (Mahomed et al., “Rates and Outcomes of Primary andRevision Total Hip Replacement in the United States MedicarePopulation,” J. Bone Joint Surg. Am. 85(A-1):27-32 (2003); WHO GlobalStrategy for Containment of Antimicrobial Resistance, 2001). Theseinfections require a very expensive two-stage revision surgery, andrecent reports suggest that success rates could be as low as 50% (Azzamet al., “Outcome of a Second Two-stage Reimplantation for PeriprostheticKnee Infection,” Clin. Orthop. Relat. Res. 467(7):1706-14 (2009)).However, the greatest concern is the emergence of drug resistantstrains, most notably MRSA, which has surpassed HIV as the most deadlypathogen in North America, and continues to make the management ofchronic OM more difficult, placing a great demand for novel therapeuticinterventions. There is a great need for alternative interventionalstrategies, particularly for immune compromised elderly who are theprimary recipients of TJR.

Presently, there are no prophylactic treatments that can protecthigh-risk patients from MRSA, most notably the aging “baby boomers” whoaccount for most of the 1.5 million TJR performed annually in the UnitedStates. A vaccine that would decrease the MRSA incidence by 50-80% wouldnot only reduce the number one complication of joint replacement andopen fracture repair procedures, but also cut the healthcare burden by asimilar amount.

Studies have documented that 80% of chronic OM is caused by S. aureus.These bacteria contain several factors that make them bone pathogensincluding several cell-surface adhesion molecules that facilitate theirbinding to bone matrix (Flock et al., “Cloning and Expression of theGene for a Fibronectin-Binding Protein From Staphylococcus aureus,”Embo. J. 6(8):2351-7 (1987)), toxins capable of stimulating boneresorption (Nair et al., “Surface-Associated Proteins FromStaphylococcus aureus Demonstrate Potent Bone Resorbing Activity,” J.Bone Miner. Res. 10(5):726-34 (1995)), through increased osteoclastactivity (Marriott et al., “Osteoblasts Express the InflammatoryCytokine Interleukin-6 in a Murine Model of Staphylococcus aureusOsteomyelitis and Infected Human Bone Tissue,” Am. J. Pathol.164(4):1399-406 (2004)). The rate-limiting step in the evolution andpersistence of infection is the formation of biofilm around implanteddevices (Costerton et al., “Bacterial Biofilms: A Common Cause ofPersistent Infections,” Science 284(5418):1318-22 (1999)). Shortly afterimplantation, a conditioning layer composed of host-derived adhesins(including fibrinogen, fibronectin, and collagen) forms on the surfaceof the implant and invites the adherence of free-floating bacteriaderived from hematogenous seeding, including spread of infection from acontiguous area (the skin adjacent to a wound), surgical inoculation ofbacteria into bone, or trauma coincident with significant disruption ofthe associated soft tissue bone envelope (Darouiche, “Treatment ofInfections Associated With Surgical Implants,” N. Engl. J. Med.350(14):1422-9 (2004)). Over the next few days bacterial cell division,recruitment of additional planktonic organisms, and secretion ofbacterial products (such as the glycocalyx) produces the biofilm. Thisbiofilm serves as a dominant barrier to protect the bacteria from theaction of antibiotics, phagocytic cells, antibodies and impairslymphocyte functions (Gray et al., “Effect of Extracellular SlimeSubstance From Staphylococcus epidermidis on the Human Cellular ImmuneResponse,” Lancet 1(8373):365-7 (1984); Johnson et al., “InterferenceWith Granulocyte Function By Staphylococcus epidermidis Slime,” Infect.Immun. 54(1):13-20 (1986); Naylor et al., “Antibiotic Resistance ofBiomaterial-Adherent Coagulase-Negative and Coagulase-PositiveStaphylococci,” Clin. Orthop. Relat. Res. 261:126-33 (1990)).

Another recent discovery is that S. aureus not only colonizes bonematrix, but is also internalized by osteoblasts in vitro (Ellington etal., “Involvement of Mitogen-Activated Protein Kinase Pathways inStaphylococcus aureus Invasion of Normal Osteoblasts,” Infect. Immun.69(9):5235-42 (2001)) and in vivo (Reilly et al., “In VivoInternalization of Staphylococcus aureus by Embryonic ChickOsteoblasts,” Bone 26(1):63-70 (2000)). This provides yet another layerof antibody and antibiotic resistance. This phase of infection occursunder conditions of markedly reduced metabolic activity and sometimesappears as so-called small-colony variants that likely accounts for itspersistence (Proctor et al., “Persistent and Relapsing InfectionsAssociated with Small-Colony Variants of Staphylococcus aureus,” Clin.Infect. Dis. 20(1):95-102 (1995)). At this point the bacteria may alsoexpress phenotypic resistance to antimicrobial treatment, alsoexplaining the high failure rate of short courses of therapy (Chuard etal., “Resistance of Staphylococcus aureus Recovered From InfectedForeign Body in Vivo to Killing by Antimicrobials,” J. Infect. Dis.163(6):1369-73 (1991)). Due to these extensive pathogenic mechanism, OMis notorious for its tendency to recur even after years of quiescence,and it is accepted that a complete cure is an unlikely outcome (Maderand Calhoun, “Long-Bone Osteomyelitis Diagnosis and Management,” Hosp.Pract. (Off Ed) 29(10):71-6, 9, 83 passim (1994)).

One of the key questions in the field of chronic OM is why currentknowledge of factors that regulate chronic OM so limited. Supposedly,the experimental tools necessary to elucidate bacterial virulence genehave been available for over a century. There are three explanations forthis anomaly. First, although the total number of osteomyelitis cases ishigh, its incidence of 1-5% is too low for rigorous prospective clinicalstudies, with the possible exception of revision arthropasty. Second, itis well known that in vitro cultures rapidly select for growth oforganisms that do not elaborate an extracellular capsule, such thatbiofilm biology can only be studied with in vivo models (Costerton etal., “Bacterial Biofilms: A Common Cause of Persistent Infections,”Science 284(5418):1318-22 (1999)). This leads to the “greatest obstacle”in this field, which is the absence of a quantitative animal model thatcan assess the initial planktonic growth phase of the bacteria prior tobiofilm formation. To date, much of the knowledge of its pathogenesiscomes from animal models (Norden, “Lessons Learned From Animal Models ofOsteomyelitis,” Rev. Infect. Dis. 10(1):103-10 (1988)), which have beendeveloped for the chicken (Daum et al., “A Model of Staphylococcusaureus Bacteremia, Septic Arthritis, and Osteomyelitis in Chickens,” J.Orthop. Res. 8(6):804-13 (1990)), rat (Rissing et al., “Model ofExperimental Chronic Osteomyelitis in Rats,” Infect. Immun. 47(3):581-6(1985)), guinea pig (Passl et al., “A Model of ExperimentalPost-Traumatic Osteomyelitis in Guinea Pigs,” J. Trauma 24(4):323-6(1984)), rabbit (Worlock et al., “An Experimental Model ofPost-Traumatic Osteomyelitis in Rabbits,” Br. J. Exp. Pathol.69(2):235-44 (1988)), dog (Varshney et al., “Experimental Model ofStaphylococcal Osteomyelitis in Dogs,” Indian J. Exp. Biol. 27(9):816-9(1989)), sheep (Kaarsemaker et al., “New Model for Chronic OsteomyelitisWith Staphylococcus aureus in Sheep,” Clin. Orthop. Relat. Res.339:246-52 (1997)) and most recently mouse (Marriott et al.,“Osteoblasts Express the Inflammatory Cytokine Interleukin-6 in a MurineModel of Staphylococcus aureus Osteomyelitis and Infected Human BoneTissue,” Am. J. Pathol. 164(4):1399-406 (2004)). While these models havebeen used to confirm the importance of bacterial adhesions identifiedfrom in vitro assays (Chuard et al., “Susceptibility of Staphylococcusaureus Growing on Fibronectin-Coated Surfaces to BactericidalAntibiotics,” Antimicrob. Agents Chemother. 37(4):625-32 (1993); Buxtonet al., “Binding of a Staphylococcus aureus Bone Pathogen to Type ICollagen,” Microb. Pathog. 8(6):441-8 (1990); Switalski et al., “ACollagen Receptor on Staphylococcus aureus Strains Isolated FromPatients With Septic Arthritis Mediates Adhesion to Cartilage,” Mol.Microbiol. 7(1):99-107 (1993)), they do not have an outcome measure ofin vivo growth, bacterial load, or osteolysis. Thus, they cannot beefficiently used to assess drug effects, bacterial mutants, and the roleof host factors with transgenic mice.

Based on over 150 years of research, a clear paradigm to explainmicrobial pathogenesis has emerged. This model also applies to OM. Theinitial step of infection occurs when a unicellular bacterium invadesthe body. At this point the microbe must respond to environmentalchanges and express virulence genes that will help it defeat innateimmunity and provide it with adhesin receptors to attach to the host.The bacterium is also dependent on the stochastic availability of hostadhesins from necrotic tissue or a foreign body such as an implant.Successful completion of these steps leads to an exponential growthphase, which ceases at the point of nutrient exhaustion and/or thedevelopment of adaptive immunity. Following the exponential growth phasethe bacteria are forced to persist under dormant growth conditionswithin the biofilm. However, at this point the infection is now chronicand cannot be eradicated by drugs or host immunity. Thus, the focus inthis field has been on cell surface adhesins that specifically interactwith extracellular matrix components known as MSCRAMMs (microbialsurface components recognizing adhesive matrix molecules) (Patti et al.,“MSCRAMM-Mediated Adherence of Microorganisms to Host Tissues,” Annu.Rev. Microbiol. 48:585-617 (1994)). In fact, essentially all anti-S.aureus vaccines that have been developed to date have been directedagainst MSCRAMMs that are important for host tissue colonization andinvasion. The goal of these vaccines is to generate antibodies that bindto these surface antigens, thereby inhibiting their attachment to hosttissue. By opsinizing the bacterial surface, these antibodies can alsomediate S. aureus clearance by phagocytic cells. Unfortunately, S.aureus has many adhesins, such that inhibition of one or more may not besufficient to prevent bacterial attachment. Furthermore, bacterialclearance by phagocytic cells may be limited in avascular tissue, suchthat mAb may need additional anti-microbial mechanism of action tosignificantly reduce the in vivo planktonic growth of S. aureus andprevent the establishment of chronic OM or reinfection during revisiontotal joint replacement surgery.

The present invention is directed to overcoming these and otherdeficiencies in the art.

SUMMARY OF THE INVENTION

A first aspect of the present invention is directed to a monoclonalantibody or binding portion thereof that binds specifically to aStaphylococcus aureus glucosaminidase and inhibits in vivo growth of S.aureus. In one embodiment, the monoclonal antibody or binding portionthereof binds specifically to an epitope lying wholly or partly withinan R3 domain of the S. aureus glucosaminidase.

A second aspect of present invention relates to a cell line thatexpresses a monoclonal antibody or binding portion of the presentinvention. In one embodiment, the cell line is a hybridoma cell line. Inanother embodiment, the cell line is a recombinant cell line thatexpresses the antibody.

A third aspect of the present invention relates to a pharmaceuticalcomposition that may include a carrier and one or more monoclonalantibodies or binding portions of the present invention.

A fourth aspect of the present invention relates to a method of treatingS. aureus infection that may include administering to a patient having aS. aureus infection an effective amount of a monoclonal antibody,binding portion, or pharmaceutical composition of the present invention.

A fifth aspect of the present invention relates to a method of treatingosteomyelitis that may include administering to a patient having a S.aureus bone or joint infection an effective amount of a monoclonalantibody, binding portion, or pharmaceutical composition of the presentinvention.

A sixth aspect of the present invention relates to a method ofintroducing an orthopedic implant into a patient that may includeadministering to a patient in need of an orthopedic implant an effectiveamount of a monoclonal antibody, binding portion, or pharmaceuticalcomposition of the present invention, and introducing the orthopedicimplant into the patient. In this aspect of the present invention, themonoclonal antibody, binding portion, or pharmaceutical composition actsas a prophylactic agent. In certain embodiments, this aspect of theinvention is directed to preventing OM or S. aureus reinfection duringor subsequent to revision total joint replacement surgery.

Because S. aureus, and especially antibiotic resistant variants such asmethicillin resistant S. aureus (MRSA), are the most common andchallenging causes of Staphylococcus infections, the methods of thepresent invention aim to disrupt critical steps in the growth cycle ofthese microorganisms. The present invention also relates to a passiveimmunization for preventing infections in patients, for example,patients undergoing total joint replacement. The selected target forimmunization is the glucosaminidase (Gmd) that S. aureus secretes tofacilitate cytokinesis, the separation of cells during mitosis (Oshidaet al., “A Staphylococcus aureus Autolysin that has anN-acetylmuramoyl-L-Alanine Amidase Domain and anEndo-beta-N-acetylglucosaminidase Domain: Cloning, Sequence Analysis,and Characterization,” Proc Natl Acad Sci USA 92:285-9 (1995); Oshida etal., “Expression Analysis of the Autolysin Gene (atl) of Staphylococcusaureus,” Microbiol Immunol 42:655-9 (1998); Sugai et al., “LocalizedPerforation of the Cell Wall by a Major Autolysin: atl Gene Products andthe Onset of Penicillin-induced Lysis of Staphylococcus aureus,” JBacteriol 179:2958-62 (1997); and Yamada et al., “An Autolysin RingAssociated with Cell Separation of Staphylococcus aureus,” J Bacteriol178:1565-71 (1996), which are hereby incorporated by reference in theirentirety).

To study and evaluate S. aureus infections, OM and various therapiesdirected towards Staphylococcus infections, a novel murine model ofimplant-associated OM in which a stainless steel pin is coated with S.aureus and implanted transcortically through the tibial metaphysic wasused (Li et al., “Quantitative Mouse Model of Implant-AssociatedOsteomyelitis and the Kinetics of Microbial Growth, Osteolysis, andHumoral Immunity,” J. Orthop. Res. 26(1):96-105 (2008), which is herebyincorporated by reference in its entirety). This model provides highlyreproducible OM with Gram-positive biofilm, osteolysis,sequestrum/involucrum formation, and closely resembles clinical OM.Furthermore, in vivo bioluminescence imaging was used to quantify theplanktonic growth phase of the bacteria; real time quantitative-PCR(RTQ-PCR) was used to determine nuc gene copy number in infected bonetissue to quantify the total bacteria load; and micro-CT was used toquantify osteolysis.

Using the above-mentioned murine model of osteomyelitis, antibodiesspecific for Gmd have been identified as a conspicuous part of thesuccessful immune response in the challenged mice. In addition, avaccine comprising recombinant Gmd with N-terminal His₆ (Gmd-His)elicited at least partial immunity in the mouse model. The anti-Gmdantibodies can block S. aureus cell division by either directly blockingcell division or by recruiting host effectors such as phagocytes orcomplement at a vulnerable point in the cycle of cell division.

Experiments demonstrating the action of individual monoclonal antibodieson the cell growth of S. aureus are presented in detail in theaccompanying examples. The specific objective was to determine if singleantibodies, in the absence of any immune effectors, would suppress oralter the growth of rapidly dividing S. aureus. The growth-relatedincrease in light-scattering by growing cultures of S. aureus Xen29 wasreduced by five selected monoclonal antibodies, but they did not appearto actually alter the in vitro growth rate per se. Rather, they appearto have reduced the activity of Gmd to a degree such that dividing cellsfailed to separate from each other. The effect was dose-dependent andconsistent with a high affinity interaction between each antibody andGmd. These effects demonstrate that these antibodies, raised againstrecombinant Gmd, react effectively with native Gmd and diminish itsenzymatic activity. One of the monoclonal antibodies, 1C11, demonstratedthe unique ability to promote cell-independent lysis of S. aureus, andtwo monoclonal antibodies, 1C11 and 3A8, demonstrated an ability toinhibit in vivo S. aureus growth and infection during orthopedic implantsurgery in an in vivo mouse model.

Accordingly, it is an object of the invention to not encompass withinthe invention any previously known product, process of making theproduct, or method of using the product such that Applicants reserve theright and hereby disclose a disclaimer of any previously known product,process, or method. It is further noted that the invention does notintend to encompass within the scope of the invention any product,process, or making of the product or method of using the product, whichdoes not meet the written description and enablement requirements of theUSPTO (35 U.S.C. §112, first paragraph) or the EPO (Article 83 of theEPC), such that Applicants reserve the right and hereby disclose adisclaimer of any previously described product, process of making theproduct, or method of using the product.

It is noted that in this disclosure and particularly in the claimsand/or paragraphs, terms such as “comprises”, “comprised”, “comprising”and the like can have the meaning attributed to it in U.S. Patent law;e.g., they can mean “includes”, “included”, “including”, and the like;and that terms such as “consisting essentially of” and “consistsessentially of” have the meaning ascribed to them in U.S. Patent law,e.g., they allow for elements not explicitly recited, but excludeelements that are found in the prior art or that affect a basic or novelcharacteristic of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-C show the quantification of osteolysis from implant-associatedosteomyelitis. A longitudinal series of X-rays from a representativemouse demonstrate the development of implant-associated osteolysis overtime in this model (FIG. 1A). Medial views of reconstructed μCT(micro-computed tomography) images of representative tibiae from mice(N=5) that received a trans-tibial pin coated with S. aureus and weresacrificed on the indicated day (FIG. 1B). Also shown are control micethat received a trans-tibial pin coated with S. aureus and treated withparenteral gentamicin (Gent), or received a sterile pin. The osteolyticarea around the pin was quantified as previously described (Li et al.,“Quantitative Mouse Model of Implant-Associated Osteomyelitis and theKinetics of Microbial Growth, Osteolysis, and Humoral Immunity,” J.Orthop. Res. 26(1):96-105 (2008), which is hereby incorporated byreference in its entirety), and the data are presented as the mean+/−SD(* p<0.05 vs. Day 4; ** p<0.05 vs. Gent Day 18) (FIG. 1C). There was nodifference in the osteolysis area between the gentamicin and sterile pincontrols.

FIGS. 2A-H show the histology of trans-tibial implant-associated OM. H&E(Haematoxylin and Eosin stain) (FIGS. 2A-C), TRAP (Tartrate-ResistantAcid Phosphatase) (FIGS. 2D-F) and Gram stained (FIGS. 3G and 3H)sections of histology at the pin site (*) adjacent to the tibial cortex(#), 9 days after implantation of a sterile pin (FIGS. 2A, 2D, and 2G),or a pin coated with S. aureus (FIGS. 2B, 2C, 2E, 2F, and 2H). Of noteis the new bone (h) that forms around the sterile pin (FIG. 2A, 2D, and2G) vs. the necrotic sequestrum (s) and involucrum (i) adjacent to theinfected pin. While very few TRAP+ osteoclasts (yellow arrow heads) werepresent in the uninfected samples (FIG. 2D), numerous osteoclasts appearto be actively resorbing the cortex adjacent to the infected pin, andremodeling the new woven bone that is encasing the involucrum (FIGS. 2Eand 2F). Gram staining confirmed the absence of bacteria in thespecimens with the sterile pin (FIG. 2G) and their presence (black arrowheads) within the necrotic bone around the infected pins.

FIGS. 3A-C show the inverse correlation between bacterial load andhumoral immunity against S. aureus antigens during the establishment ofchronic osteomyelitis. A time course study was performed in which micewere given an infected transcortical pin containing1×10⁶ CFU of S.aureus in their tibia and sacrificed on the indicated day. At sacrifice,DNA was purified from the infected tibia and RTQ-PCR was performed todetermine the Ct values for S. aureus nuc. Using a standard curve shown,this number was converted to the recoverable nuc genes per tibia. Tocontrol for the integrity of the samples, the recoverable nuc gene pertibia value was standardized to the Ct value for mouse β-actin for eachsample. From this conversion the bacterial load was derived as “Nuc GeneCopies/Tibia.” The data from each mouse is shown in FIG. 3A as anindividual point, and the mean+/−SD for each time point (n=5) ispresented in FIG. 3B. To assess the development of anti-S. aureusspecific antibodies during the establishment of OM, serum was taken fromeach mouse in the group that was sacrificed on day 18, before infection(day 0) and on days 4, 7, 11, 14 and 18 after infection. This serum wasused as the primary antibody in Western blots of total S. aureus extractthat were then probed with HRP-conjugated antibodies that are specificfor mouse IgG as shown in FIG. 3C. The data show that there is a steadyincrease in bacterial growth from day 0 to day 11, when the host firstdevelops specific antibodies against the bacteria. As the titer of theanti-S. aureus antibodies increases the bacterial load drops, suggestingthat the antibodies are protective. The Western blots also clearlyidentify four immuno-dominant antigens of 26, 34, 38 and 56 kDa(arrows). It has also been demonstrated that Xen 29 also inducesantibodies against these same 26, 34, 38 and 56 kDa proteins.

FIGS. 4A-C show that glucosaminidase of S. aureus autolysin is the 56kDa immuno-dominant antigen. To elucidate the molecular identity of thenovel S. aureus antigens identified in FIG. 3, subtractive immunoblotanalysis of 2D-SDS-PAGE of whole cell extract was performed withpre-immune and day 14 immune sera. Three 2D gels were run afterisoelectric focusing (pH 4.0-10.0). The first was Coomassie blue-stained(FIG. 4A). The others were Western blotted with either day 0 (FIG. 4B)or day 14 sera (FIG. 4C). In addition to the background reactivity, theimmune serum detected a specific polypeptide (˜53 kDa; pH 9: arrow). The53 kDa spot was removed from the Coomassie gel, digested with trypsin,and analyzed by MALDI, which resolved 70 individual peptide peaks. Theamino acid sequence from every peptide was a 100% match with the knownsequence of the glucosaminidase of S. aureus autolysin, which is 53.6kDa and has a pI of 9.66.

FIGS. 5A-B show bioluminescent imaging (BLI) quantification of bacterialgrowth during the establishment of chronic osteomyelitis. FIG. 5A showsBLI levels (p/sec/cm²/sr) at the site of infection and was assessedlongitudinally in mice that received a sterile trans-tibial pin(Uninfected), or a pin coated with Xen 29 S. aureus (Infected) and wereimaged on the indicated day. The circle in the top left image highlightsthe 1.5 cm diameter region of interest (ROI) that was assessed for BLIin each mouse at each time point. FIG. 5B shows the data from mice (N=5)that were Uninfected, Infected or infected and treated with parenteralantibiotics (Gentamycin) and were assessed for BLI longitudinally at theindicated time following surgery. The data are presented as themean+/−SD (* Significantly greater vs. Day 0; p<0.05).

FIGS. 6A-B show that functional anti-Gmd ELISA demonstrated the efficacyof recombinant Gmd vaccine. FIG. 6A shows serum ELISA in which His-Gmdwas used as the antigen to assay anti-Gmd antibody titers in mouse serumwhich was generated using a known high titer anti-sera from S. aureusinfected mice. The serial dilution factor (X axis) and absorbancereading at 450 nm (Y axis) of the serial 2-fold diluted sera samples areplotted in the XY plane using GraphPad Prism 4 software. The functionaltiter (1:3623) is extrapolated from the inflection point (arrow) of thedilution curve. FIG. 6B shows the ELISA used to determine the titers ofanti-Gmd antibodies in the sera of mice pre-immunization, pre-boost andpre-challenge with the indicated vaccine. Note that only mice immunizedwith the His-Gmd vaccine obtained high titers.

FIGS. 7A-C show that recombinant His-Gmd vaccine protects mice fromimplant-associated OM. The mice (n=20) were challenged with a Xen29infected transtibial pin as described in the accompanying Examples, BLIwas performed on day 3, and the mice were euthanized for nuc RTQ-PCR onday 11. An image of the BLI from a representative mouse in Group 1 & 3is shown (FIG. 7A), and the mean+/−SD is presented to show thesignificant reduction BLI (FIG. 7B). This translated into a significantdecrease in amplifiable nuc genes (mean+/−SD) on day 11 (FIG. 7C).

FIG. 8 shows the in vitro growth of S. aureus Xen29 in the presence ofanti-Gmd monoclonal antibodies (anti-Gmd mAb). 100 cfu of Xen29 from aculture in log-phase growth were incubated at 37° C. with anti-Gmdmonoclonal antibodies 1C11, 1E12, 2D11, and 3A8, 50 μg/mL in LB medium.Growth was monitored by light scattering at both 670 and 490 nm at theindicated intervals. MOPC21 is the isotype-matched control antibody.

FIG. 9 shows the dose-dependent effect of anti-Gmd mAb 1C11 on in vitroS. aureus growth. 100 cfu of Xen29 from a culture in log-phase growthwere incubated at 37° C. with a range of concentrations of anti-Gmd mAb1C11 in LB medium. Growth was monitored by light scattering at both 670and 490 nm at the indicated intervals.

FIG. 10 shows the effect of Control mAb MOPC21 on in vitro S. aureusgrowth. 100 cfu of Xen29 from a culture in log-phase growth wereincubated at 37° C. with a range of concentrations of isotype-matchedcontrol monoclonal antibody MOPC21 in LB medium. Growth was monitored bylight scattering at both 670 and 490 nm at the indicated intervals. Notethat the slight elevation of the 50 μg/mL line is due to the use ofoutside wells on the microtiter plate where temperatures equilibratefaster.

FIG. 11 shows a ClustalW amino acid sequence alignment of the V_(H)sequences from hybridomas 2D11, 3H6, 1E12 and 3A8. (2D11 V_(H)=SEQ IDNO: 1; 3H6 V_(H)=SEQ ID NO: 2; 1E12 V_(H)=SEQ ID NO: 3; 3A8 V_(H)=SEQ IDNO: 4). Highlighted sequences indicate the putative complementaritydetermining regions (CDR) in 2D11. A consensus sequence (SEQ ID NO: 31)is derived from these hybridoma sequences.

FIG. 12 shows a ClustalW amino acid sequence alignment of V_(L)sequences from hybridomas 1E12 and 2D11 (1E12 V_(L)=SEQ ID NO: 10; 2D11V_(L)=SEQ ID NO: 8). A consensus sequence (SEQ ID NO: 32) is derivedfrom these two hybridoma sequences.

FIG. 13 shows a ClustalW amino acid sequence alignment of the V_(H)sequences from hybridomas 2D11, 3H6, 1E12, 3A8, and 1C11 (2D11 V_(H)=SEQID NO: 1; 3H6 V_(H)=SEQ ID NO: 2; 1E12 V_(H)=SEQ ID NO: 3; 3A8 V_(H)=SEQID NO: 4; 1C11 V_(H)=SEQ ID NO: 5). A consensus sequence (SEQ ID NO: 6)is derived from these five hybridoma sequences.

FIGS. 14A-B show a ClustalW amino acid sequence alignment of the V_(L)sequences. FIG. 14A shows V_(L) alignment of hybridomas 1E12, 2D11, 3A8,3H6, and 1C11 (1E12 V_(L)=SEQ ID NO: 10; 2D11 V_(L)=SEQ ID NO: 8; 3A8V_(L)=SEQ ID NO: 11; 3H6 V_(L)=SEQ ID NO: 9; 1C11 V_(L)=SEQ ID NO: 12).A consensus sequence (SEQ ID NO: 13) is derived from these fivehybridoma sequences. FIG. 14B shows V_(L) alignment of hybridomas 1E12,2D11, 3A8, and 3H6 (1E12 V_(L)=SEQ ID NO: 10; 2D11 V_(L)=SEQ ID NO: 8);3A8 V_(L)=SEQ ID NO: 11; 3H6 V_(L)=SEQ ID NO: 9). A consensus sequence(SEQ ID NO: 33) is derived from these four hybridoma sequences.

FIG. 15 shows a ClustalW amino sequence alignment of the V_(H) sequencesfrom hybridomas 3A8 and 1C11 (3A8 V_(H)=SEQ ID NO: 4; 1C11 V_(H)=SEQ IDNO: 5). A consensus sequence (SEQ ID NO: 7) is derived from these twohybridoma sequences.

FIG. 16 shows a ClustalW amino acid sequence alignment of the V_(L)sequences from hybridomas 3A8 and 1C11 (3A8 V_(L)=SEQ ID NO: 11; 1C11V_(L)=SEQ ID NO: 12). A consensus sequence (SEQ ID NO: 14) is derivedfrom these two hybridoma sequences.

FIG. 17A illustrates an alignment of the 1C11 V_(H) domain (SEQ ID NO:5) with a homologous amino acid sequence (SEQ ID NO: 19) encoded by thehuman gene IGV7-81 (see Genbank Accession AAH32733 and BC032733, each ofwhich is hereby incorporated by reference in its entirety). A consensussequence for the V_(H) homologs (SEQ ID NO: 34) is shown. FIG. 17Billustrates an alignment of the 1C11 V_(L) domain (SEQ ID NO: 12) with ahomologous amino acid sequence (SEQ ID NO: 20) encoded by the human geneIGVK6D-21 (see Genbank Accession AAA58917 and M29469, each of which ishereby incorporated by reference in its entirety). A consensus sequencefor the V_(L) homologs (SEQ ID NO: 35) is shown.

FIG. 18 shows the inhibition of S. aureus His-Gmd (Gmd) and hen egglysozyme (HEL) by the five anti-Gmd monoclonal antibodies with MOPC21 asan isotype-matched negative control. The concentration of the antibodyin μg/mL is listed on the x-axis; the inhibition of enzyme activity inpercentage (%) is listed on the Y-axis. All five anti-Gmd mAbs (1C11,1E12, 2D11, 3A8, and 3H6) inhibit Gmd activity, but have no effect onHEL activity, and MOPC21 (negative control) does not inhibit eitherenzyme.

FIG. 19 shows inhibition of native Gmd by the five anti-His-Gmd mAbs1C11, 1E12, 2D11, 3A8, and 3H6. Each antibody was added at aconcentration of 100 μg/mL. All five are potent inhibitors and inhibitthe native enzyme to about the same degree as they inhibit therecombinant Gmd-His. The isotype-matched (IgG1) antibody control MOPC21had no effect on Gmd enzymatic activity.

FIGS. 20A-D are scanning electron miscroscopy (SEM) images of S. aureusgrown in the absence (FIGS. 20A-B) or presence of anti-Gmd monoclonalantibodies of the present invention. FIG. 20C shows the effects of 50μg/ml mAb 1E12 on Xen29 S. aureus and FIG. 20D shows the effects of 50μg/ml mAb 1C11 on Xen29 S. aureus. Micrographs of representative fieldswere obtained at 50,000× (A&C), 2,000× (B) and 4,000× (D). Arrowsidentify sites where lysis has occurred, and document the surprising andunexpected effects of the present invention, as complement and immuneeffector cell independent lytic activity of an anti-S. aureus mAb hasyet to be documented in the literature.

FIGS. 21A-C show that passive immunization with monoclonal antibody 3A8inhibits S. aureus growth in vivo and protects mice fromimplant-associated osteomyelitis. The mice were imaged to assessbioluminescence on days 0, 3, 5, 7, 11, and 14, and images with the BLIheat map from a representative animal in each group are shown in FIG.21A. The BLI values on day 3 for each mouse in the study are shown withthe mean for each group (FIG. 21B, p=0.02). X-rays from a representativeanimal in each group obtained on day 14 are shown to illustrate theosteolytic lesion (arrow) in the placebo mouse, which was not present inthe anti-Gmd treated animals (FIG. 21C).

FIGS. 22A-C show that passive immunization with monoclonal antibody 1C11inhibits S. aureus growth in vivo and protects mice fromimplant-associated osteomyelitis. The mice were imaged to assessbioluminescence on days 0, 3, 5, 7, 10, and 14, and images with the BLIheat map from a representative animal in each group are shown in (FIG.22A). The BLI values on day 3 for each mouse in the study are shown withthe mean for each group (FIG. 22B). X-rays from a representative animalin each group obtained on day 14 are shown to illustrate the osteolyticlesion (arrow) in the placebo mouse, which was not present in theanti-Gmd treated mouse (FIG. 22C).

FIG. 23 is a graph comparing the anti-Gmd inhibitory activity of mousemonoclonal 1C11 with the humanized chimeric monoclonal derived from1C11. The human:mouse chimeric IgG1 of 1C11 (h1C11) retains the abilityto inhibit His-Gmd. The percent inhibition of His-Gmd activity on theY-axis is displayed as a function of dilution of the antibodypreparation on the X-axis. The mouse 1C11 concentration was 10 μg/mL;the concentration for chimeric h1C11 was not known for the assay shown.

DETAILED DESCRIPTION OF THE INVENTION

In one aspect, the present invention relates to a monoclonal antibodythat binds specifically to a Staphylococcus aureus glucosaminidase andinhibits in vivo growth of S. aureus. The monoclonal antibody of thepresent invention can be such that it targets S. aureus that ismethicillin resistant.

As used herein, the term “antibody” is meant to include immunoglobulinsderived from natural sources or from recombinant sources, as well asimmunoreactive portions (i.e. antigen binding portions) ofimmunoglobulins. The monoclonal antibodies of the present invention mayexist in or can be isolated in a variety of forms including, forexample, substantially pure monoclonal antibodies, antibody fragments orbinding portions, chimeric antibodies, and humanized antibodies (EdHarlow and David Lane, USING ANTIBODIES: A LABORATORY MANUAL (ColdSpring Harbor Laboratory Press, 1999), which is hereby incorporated byreference in its entirety).

The monoclonal antibodies of the present invention are characterized byspecificity for binding to S. aureus glucosaminidase or fragmentsthereof. The antibody specifically binds to an immuno-dominant epitopein the glucosaminidase (Gmd) sub-unit of S. aureus autolysin (Atl).These monoclonal antibodies inhibit in vivo growth of S. aureus.

Immuno-dominant antigen is a part of the antigenic determinant that ismost easily recognized by the immune system and thus exerts the mostinfluence on the specificity of the induced antibody. An“immuno-dominant epitope” refers to the epitope on an antigen thatselectively provokes an immune response in a host organism to thesubstantial exclusion of other epitopes on that antigen.

Usually, the antigen likely to carry an immuno-dominant epitope can beidentified by selecting antigens on the outer surface of the pathogenicorganism. For example, most simple organisms, such as fungi, bacteriaand viruses have one or two proteins that are exposed on the outersurface of the pathogenic organism. These outer surface proteins aremost likely to carry the appropriate antigen. The proteins most likelyto carry an immuno-dominant epitope can be identified in a Western assayin which total protein is run on a gel against serum from an organisminfected with the pathogenic organism. Bound antibodies from the serumare identified by labeled anti-antibodies, such as in one of thewell-known ELISA techniques. The immuno-dominant epitope can beidentified by examining serum from a host organism infected with thepathogenic organism. The serum is evaluated for its content ofantibodies that bind to the identified antigens that are likely to causean immune response in a host organism. If an immuno-dominant epitope ispresent in these antigens, substantially all antibodies in the serumwill bind to the immuno-dominant epitope, with little binding to otherepitopes present in the antigen.

Atl is one of the catalytically distinct peptidoglycan hydrolases in S.aureus that is required to digest the cell wall during mitosis (Baba andSchneewind, “Targeting of Muralytic Enzymes to the Cell Division Site ofGram-Positive Bacteria: Repeat Domains Direct Autolysin to theEquatorial Surface Ring of Staphylococcus aureus, ” EMBO. J.17(16):4639-46 (1998), which is hereby incorporated by reference in itsentirety). In addition to being an essential gene for growth, scanningelectron microscopy studies have demonstrated that anti-Atl antibodiesbound to S. aureus during binary fission localize to regions of thebacteria that are not covered by the cell wall (Yamada et al., “AnAutolysin Ring Associated With Cell Separation of Staphylococcusaureus,” J. Bacteria 178(6):1565-71 (1996), which is hereby incorporatedby reference in its entirety).

The Atl enzyme is comprised of an amidase (62 kD) and glucosaminidase(53 kD), which are produced from the same Atl precursor protein via acleavage process (Baba and Schneewind, “Targeting of Muralytic Enzymesto the Cell Division Site of Gram-Positive Bacteria: Repeat DomainsDirect Autolysin to the Equatorial Surface Ring of Staphylococcusaureus,” Embo. J. 17(16):4639-46 (1998); Komatsuzawa et al.,“Subcellular Localization of the Major Autolysin, ATL and Its ProcessedProteins in Staphylococcus aureus,” Microbiol Immunol. 41:469-79 (1997);Oshida et al., “A Staphylococcus aureus Autolysin That Has anN-acetylmuramoyl-L-alanine Amidase Domain and anEndo-beta-N-acetylglucosaminidase Domain: Cloning, Sequence Analysis,and Characterization,” Proc. Nat'l. Acad. Sci. U.S.A. 92(1):285-9(1995), which are hereby incorporated by reference in their entirety).The autolysin is held to the cell wall by three ˜150 amino acid cellwall binding domains R1, R2, and R3. In the final maturation step,proteolytic cleavage separates the aminidase domain and its associatedR1 and R2 domains from the glucosaminidase and its associated N-terminalR3 domain.

By way of example, and without limitation, one exemplary Staphylococcusaureus glucosaminidase contains the amino acid sequence of SEQ ID NO: 36below.

AYTVTKPQTT QTVSKIAQVK PNNTGIRASV YEKTAKNGAK YADRTFYVTK ERAHGNETYV LLNNTSHNIP LGWFNVKDLNVQNLGKEVKT TQKYTVNKSN NGLSMVPWGT KNQVILTGNNIAQGTFNATK QVSVGKDVYL YGTINNRTGW VNAKDLTAPTAVKPTTSAAK DYNYTYVIKN GNGYYYVTPN SDTAKYSLKAFNEQPFAVVK EQVINGQTWY YGKLSNGKLA WIKSTDLAKE LIKYNQTGMT LNQVAQIQAG LQYKPQVQRV PGKWTDANFNDVKHAMDTKR LAQDPALKYQ FLRLDQPQNI SIDKINQFLKGKGVLENQGA AFNKAAQMYG INEVYLISHA LLETGNGTSQLAKGADVVNN KVVTNSNTKY HNVFGIAAYD NDPLREGIKYAKQAGWDTVS KAIVGGAKFI GNSYVKAGQN TLYKMRWNPA VHPGTHQYATD DWANINAKI IKGYYDKIGE VGKYFDIPQYIn SEQ ID NO: 36, underlined residues correspond to residues 783 to 931of the encoded autolysin, and represent the R3 domain. The remainingC-terminal residues (not underlined) correspond to the catalyticglucosaminidase domain.

In certain embodiments the monoclonal antibody of the present inventionbinds to a conserved epitope of Staphylococcus aureus glucosaminidasewith an affinity greater than 10⁻⁹ M. As used herein, “epitope” refersto the antigenic determinant of Staphylococcus aureus glucosaminidasethat is recognized by the monoclonal antibody. The epitope recognized bythe antibody of the present invention may be a linear epitope, i.e. theprimary structure of the amino acid sequence of glucosaminidase.Alternatively, the epitope recognized by the antibody of the presentinvention may be a non-linear or conformational epitope, i.e. thetertiary structure of glucosaminidase.

In certain embodiments, the monoclonal antibodies may bind specificallyto the catalytic domain of the Gmd. In other embodiments, the monoclonalantibodies may bind specifically to the R3 domain.

Epitopes that are bound by five of the monoclonal antibodies identifiedherein lie wholly or at least partially within the R3 domain. By way ofexample, an epitope bound by mAb 3A8 lies within the region containingresidues 776-842; an epitope bound by mAb 1C11 lies within the regioncontaining residues 842-873; an epitope(s) bound by mAbs 2D11 and 1E12are the same or different and lie within the region containing residues842-948; and an epitope bound by mAb 3H6 lies within the regioncontaining residues 907-948.

In certain embodiments, the monoclonal antibody of the present inventionpossesses S. aureus Gmd inhibitory activity, whereby the monoclonalantibody inhibits the activity of Gmd by at least 20%, at least 30%, atleast 40% or at least 50%. In other embodiments, the monoclonal antibodyinhibits the activity of Gmd by at least 60%, at least 70%, or at least80%. Five monoclonal antibodies described herein (mAbs 3A8, 1C11, 2D11,1E12, and 3H6) possess anti-Gmd inhibitory activity of about 70 to about80 percent. It is a surprising and unexpected result that the antibodiesof the present invention would bind a purported cell wall bindingdomain, the R3 domain, rather than a catalytic domain to inhibitenzymatic activity. Without being bound by theory, it is believed thatthe binding of the antibodies of the present invention to the R3 domainmay trigger a conformational or electrostatic change in the catalyticdomain of glucosaminidase.

Inhibition of Gmd activity can be measured in vitro. According to oneapproach, Gmd is first pre-titered to determine the concentration thatwill yield about a 50% reduction in A₄₉₀ in 60 minutes. Then 50 μL ofantibody diluted in PBST is added to each well of a 96-well microtiterplate followed by 50 μL of appropriately diluted Gmd, and the mixtureallowed to incubate for 5 or more minutes, and finally 100 μL of 0.15%mL is added and the initial A₄₉₀ measured. The plate is incubated at 37°C. and the A₄₉₀ measured at 30 and 60 minutes. Percent inhibition iscalculated as 100·(1−(Δ₆₀A₄₉₀ inhibitor/Δ₆₀A₄₉₀ no inhibitor control)).

In certain embodiments, the monoclonal antibody of the present inventionpossesses an ability to cause clustering or clumping of S. aureus,cell-independent lysis of S. aureus, or both. Examples of antibodiesthat possess an ability to cause clumping of S aureus include, withoutlimitation, monoclonal antibodies 1C11, 1E12, 2D11, 3A8, and 3H6. Oneexample of a lytic antibody is monoclonal antibody 1C11. This antibodybinds to a unique epitope present in the R3 domain, displays betweenabout 70 to about 80 percent Gmd inhibitory activity, and promotescell-independent lysis of S. aureus.

The monoclonal antibodies of the present invention also inhibit in vivogrowth of S. aureus. Inhibition of in vivo growth of S. aureus can bemeasured according to a number of suitable standards. In one suchembodiment, the in vivo growth of S. aureus can be assessed according toa bioluminescence assay of the type described in the accompanyingExamples. Specifically, bioluminescent S. aureus (Xen 29; ATCC 12600)(Francis et al., “Monitoring Bioluminescent Staphylococcus aureusInfections in Living Mice Using a Novel luxABCDE Construct,” Infect.Immun. 68(6):3594-600 (2000); see also Contag et al., “PhotonicDetection of Bacterial Pathogens in Living Hosts,” Mol. Microbiol.18(4):593-603 (1995), each of which is hereby incorporated by referencein its entirety) is used to dose a transtibial implant with 500,000 CFUprior to surgical implant. Five week old female BALB/cJ mice can receivean intraperitoneal injection of saline (n=10) or 1 mg of purifiedantibody in 0.25 ml saline 3 days prior to surgery. The mice can beimaged to assess bioluminescence on various days (e.g., 0, 3, 5, 7, 11,and 14) and a comparison of BLI images can be compared to assess whetherthe antibody inhibits in vivo growth of S. aureus relative to the salinecontrol.

In one embodiment the monoclonal antibody of the present inventioncomprises a V_(H) domain comprising one of the following amino acidsequences:

(SEQ ID NO: 1) EVQLQESGPVLVKPGASVKMSCKASGYTFTDYYMNWVKQSHGKSLEWIGVINPYNGDTTYSQKFKGKATLTVDKSSSTAYMELNSLTSEDSAVYYCARNYDEYFDVVVGTGTTVTVSSAKTTPPSVYPLAPGSAAQTNSMVT LGCXVKG; or(SEQ ID NO: 2) EVQLQESGPVLVKPGASVKLSCKASGYTFTDYFMNWVKQSHGKSLEWIGVINPFNGGNRYNQNFKGKATLTVDKSSSTAYMELNSLTSEDSAVYYCARGDYDSPWFDYWGQGTLVTVSAAKTTPPSVYPLAPGSAAQTN SMVTLGCLVKGYSXSQ,where X is any amino acid; or (SEQ ID NO: 3)EVQLQESGGGFVKPGGSLKLSCAASGFTFSTYVMSWVRQTPEKRLEWVATISDGGGHTYYLDNVKGRFTISRDNAKNNLYLHMSHLKSEDTAMYYCARAYYGSSYDAMDYWGQGTSVTVSSAKTTPPSVYPLAPGSAAQT NSMVTLGCLVKG; or(SEQ ID NO: 4) EVQLQESGGGLVQPGGSMKLSCAASGFTFSDAWMDWVRQSPEKGLEWVAEIKDKTNNHATYYAESVKGRFTISRDVSKSRVFLQMNSLRPEDTGIYYCTSGPYFDYWGQGTTLTVSSAKTTPPSVYPLAPGSAAQTNSMVT LGCLVKGYFPE; or(SEQ ID NO: 5) QIQLVQSGPELKKPGETVKISCKASGYTFTTYGMSWVNQAPGKGLKWMGWINTYSGVPTYADDFKGRFVFSLETSASTAYLQINNLKNEDTATYFCAREEYSSGYAAWFPYWGQGTLVTVSA, where X is any amino acid; orXXQLXXSGXXXXXPGXXXKXSCXASGXTFXXXXMXWVXQXXXKXLXWXXXIXXXXXXXXXXYXXXXKGXXXXXXXXXXXXXXXXXXXLXXEDXXXYXCXXXXYXXXXXXXXXXWGXGTXXTVSXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX,where X is any amino acid or deletion thereof(consensus sequence SEQ ID NO: 6; see Figure 13);  orEVQLQESGXXXVXPGXSXKXSCXASGXTFXXXXMXWVXQXXXKXLEWXXXIXXXXXXXXXXYXXXXKGXXTXXXDXXXXXXXXXXXXLXXEDXXXYYCXXXXYXXXXXXXDXWGXGTXXTVSXAKTTPP SVYPLAPGSAAQTNSMVTLGCXVKGXXXXX, where X is any amino acid or deletion thereof(consensus sequence SEQ ID NO: 31; see Figure 11),  orXXQLXXSGXXLXXPGXXXKXSCXASGXTFXXXXMXWVXQXPXKGLXWXXXIXXXTXXXXXXYAXXXKGRFXXSXXXSXSXXXLQXNXLXXEDTXXYXCXXXXXXSGXXXXFXYWGQGTXXTVSXXXXXXXXXXX XXXXXXXXXXXXXXXXXXXXXXXXX,where X is any amino acid or deletion thereof(consensus sequence SEQ ID NO: 7; see Figure 15);  orQXQLVQSGXEXKXPGXXVKXSCKASGYXFTTYGMXWVXQAPGXGLXWMGWXNTYXGXPTYAXXFXGRFVFSXXTSASTAYLQIXXLKXEDXAXYXCARXXXXXXXXXXXXYWGQGTLVTVSA,where X is any amino acid or deletion thereof(consensus sequence SEQ ID NO: 35, see Figure 17A).

In another embodiment the monoclonal antibody of the present inventioncomprises a V_(L) domain comprising one of the following amino acidsequences:

(SEQ ID NO: 8) DIVMTQSPAIMSASPGEKVTMTCSASSSVSYMYWYQQKPGSSPRLLIYDTSNLASGVPVRFSGSGSGTSYSLTISRMEAEDAATYYCQQWSSYPLT FG; or (SEQ ID NO: 9)QMTQTTSSLSASLGDRVTISCSASQGISNYLNWYQQKPDGTVKLLIYYTSSLHSGVPSRFSGGGSGTDYSLSISNLEPEDIATYYCQQYSKLPWTFG GGTKLEIK,; or(SEQ ID NO: 10) DIVITQSPAIMSASLGERVTMTCTASSSVSSSYLHWYQQKPGSSPKXWIYSTSNLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCHQYHRSPW TFGGGT; or(SEQ ID NO: 11) DIVMTQSHKFMSTSVGDRVSITCKASQDVSTAVAWYQQKPGQSPKLLIYWTSTRHTGVPDRFTGSGSGTDFTLTISSVQAKDLALYYCQQHYTTPY TFGGGTKLEIK,; or(SEQ ID NO: 12) DIVLTQSPATLSVTPGDSVSLSCRASQSISNNLHWYQQKSHESPRLLIEYASRSISGIPSRFSGGGSGTDFTLSINSVESEDFGLYFCQQSNSWPLTFGA GTKLELK,; orDIXXTQXXXXXSXXXGXXVXXXCXASXXXSXXXXXWYQQKXXXXXXXXIXXXSXXXXGXPXRFXGXGSGTXXXLXIXXXXXXDXXXYXCX QXXXXPXTFGXGTXXXXX,where X is any amino acid or deletion thereof(consensus sequence SEQ ID NO: 13, see Figure 14A); orDIVXTQSXXXXSXXXGDXVSXXCXASQXXSXXXXWYQQKXXXSPXLLIXXXSXXXXGXPXRFXGXGSGTDFTLXIXSVXXXDXXLYXCQQXXX XPXTFGXGTKLEXK,where X is any amino acid or deletion thereof(consensus sequence SEQ ID NO: 14, see Figure 16); orDIVXTQSPAIMSASXGEXVTMTCXASSSVSXXYXXWYQQKPGSSPXXXIYXTSNLASGVPXRFSGSGSGTSYSLTISXMEAEDAATYYCXQXXXX PXTFGXXX,where X is any amino acid or deletion thereof (consensus sequence SEQ ID NO: 32, see Figure 12); orDIXXTQXXXXXSXSXSXXSXXXSXSSXXXSXXXXXWYQQKPXXXXXXXIYXTSXXXXGVPPXRFXGXGSGTXXXLXISXXXXXDXAXYYCXQ XXXXPXTFGGGTXXXXX,where X is any amino acid or deletion thereof(consensus sequence SEQ ID NO: 33, see Figure 14B); orXIVLTQSPXXXSVTPXXXVXXXCRASQSIXXXLHWYQQXXXXSPXLLIXYASXSXSGXPSRFSGXGSGTDFTLXINSXEXEDXXXYXCXQSXSXPL TFGXGTKXEXK,where X is any amino acid or deletion thereof (consensus sequence SEQ ID NO: 37, see Figure 17B).

Antibodies of the present invention may also be synthetic antibodies. Asynthetic antibody is an antibody which is generated using recombinantDNA technology, such as, for example, an antibody expressed by abacteriophage. Alternatively, the synthetic antibody is generated by thesynthesis of a DNA molecule encoding and expressing the antibody of theinvention or the synthesis of an amino acid specifying the antibody,where the DNA or amino acid sequence has been obtained using syntheticDNA or amino acid sequence technology which is available and well knownin the art.

The monoclonal antibody of the present invention can be humanized.Humanized antibodies are antibodies that contain minimal sequences fromnon-human (e.g. murine) antibodies within the variable regions. Suchantibodies are used therapeutically to reduce antigenicity and humananti-mouse antibody responses when administered to a human subject. Inpractice, humanized antibodies are typically human antibodies withminimum to no non-human sequences. A human antibody is an antibodyproduced by a human or an antibody having an amino acid sequencecorresponding to an antibody produced by a human.

An antibody can be humanized by substituting the complementaritydetermining region (CDR) of a human antibody with that of a non-humanantibody (e.g. mouse, rat, rabbit, hamster, etc.) having the desiredspecificity, affinity, and capability (Jones et al., “Replacing theComplementarity-Determining Regions in a Human Antibody With Those Froma Mouse,” Nature 321:522-525 (1986); Riechmann et al., “Reshaping HumanAntibodies for Therapy,” Nature 332:323-327 (1988); Verhoeyen et al.,“Reshaping Human Antibodies: Grafting an Antilysozyme Activity,” Science239:1534-1536 (1988), which are hereby incorporated by reference intheir entirety). The humanized antibody can be further modified by thesubstitution of additional residues either in the Fv framework regionand/or within the replaced non-human residues to refine and optimizeantibody specificity, affinity, and/or capability.

Humanized antibodies can be produced using various techniques known inthe art Immortalized human B lymphocytes immunized in vitro or isolatedfrom an immunized individual that produce an antibody directed against atarget antigen can be generated (see e.g. Reisfeld et al., MONOCLONALANTIBODIES AND CANCER THERAPY 77 (Alan R. Liss ed., 1985) and U.S. Pat.No. 5,750,373 to Garrard, which are hereby incorporated by reference intheir entirety). Also, the humanized antibody can be selected from aphage library, where that phage library expresses human antibodies(Vaughan et al., “Human Antibodies with Sub-Nanomolar AffinitiesIsolated from a Large Non-immunized Phage Display Library,” NatureBiotechnology, 14:309-314 (1996); Sheets et al., “Efficient Constructionof a Large Nonimmune Phage Antibody Library: The Production ofHigh-Affinity Human Single-Chain Antibodies to Protein Antigens,” Proc.Nat'l. Acad. Sci. U.S.A. 95:6157-6162 (1998); Hoogenboom et al.,“By-passing Immunisation. Human Antibodies From Synthetic Repertoires ofGermline VH Gene Segments Rearranged in vitro,” J. Mol. Biol. 227:381-8(1992); Marks et al., “By-passing Immunization. Human Antibodies fromV-gene Libraries Displayed on Phage,” J. Mol. Biol. 222:581-97 (1991),which are hereby incorporated by reference in their entirety). Humanizedantibodies can also be made in transgenic mice containing humanimmunoglobulin loci that are capable upon immunization of producing thefull repertoire of human antibodies in the absence of endogenousimmunoglobulin production. This approach is described in U.S. Pat. No.5,545,807 to Surani et al.; U.S. Pat. No. 5,545,806 to Lonberg et al.;U.S. Pat. No. 5,569,825 to Lonberg et al.; U.S. Pat. No. 5,625,126 toLonberg et al.; U.S. Pat. No. 5,633,425 to Lonberg et al.; and U.S. Pat.No. 5,661,016 to Lonberg et al., which are hereby incorporated byreference in their entirety.

Based on a BLAST search of Genbank using the 1C11 V_(H) and V_(L) domainamino acid sequences, homologous sequences within the human genome wereidentified as IGVH7-81 and IGVK6D-21, respectively. Alignments of thesehomologous V_(H) and V_(L) domains (SEQ ID NOS: 19 and 20, respectively)with the corresponding 1C11 V_(H) and V_(L) domains are illustrated inFIGS. 17A-B, respectively. The V_(H) and V_(L) domains share asurprisingly high degree of identity, respectively about 75% and 67%over the region of homology (i.e., excluding V_(H) CDR3H region). TheseIGVH7-81 and IGVK6D-21 sequences can be used to prepare a substantiallypure monoclonal antibody of the present invention. Because the CDR3H isnot encoded by the V_(H) gene, a suitable D region will need to bespliced into the missing domain region. Any one of several candidate Dregions can be used (e.g., IGHDS-5, 18 or 12*01).

In addition to whole antibodies, the present invention encompassesbinding portions of such antibodies. Such binding portions include themonovalent Fab fragments, Fv fragments (e.g., single-chain antibody,scFv), and single variable V_(H) and V_(L) domains, and the bivalentF(ab′)₂ fragments, Bis-scFv, diabodies, triabodies, minibodies, etc.These antibody fragments can be made by conventional procedures, such asproteolytic fragmentation procedures, as described in James Goding,MONOCLONAL ANTIBODIES: PRINCIPLES AND PRACTICE 98-118 (Academic Press,1983) and Ed Harlow and David Lane, ANTIBODIES: A LABORATORY MANUAL(Cold Spring Harbor Laboratory, 1988); Houston et al., “ProteinEngineering of Antibody Binding Sites: Recovery of Specific Activity inan Anti-Digoxin Single-Chain Fv Analogue Produced in Escherichia coli,”Proc. Mad Acad. Sci. USA 85:5879-5883 (1988); Bird et al, “Single-ChainAntigen-Binding Proteins,” Science 242:423-426 (1988), which are herebyincorporated by reference in their entirety, or other methods known inthe art.

It may further be desirable, especially in the case of antibodyfragments, to modify the antibody to increase its serum half-life. Thiscan be achieved, for example, by incorporation of a salvage receptorbinding epitope into the antibody fragment by mutation of theappropriate region in the antibody fragment or by incorporating theepitope binding site into a peptide tag that is then fused to theantibody fragment at either end or in the middle (e.g., by DNA orpeptide synthesis).

Antibody mimics are also suitable for use in accordance with the presentinvention. A number of antibody mimics are known in the art including,without limitation, those known as monobodies, which are derived fromthe tenth human fibronectin type III domain (¹⁰Fn3) (Koide et al., “TheFibronectin Type III Domain as a Scaffold for Novel Binding Proteins,”J. Mol. Biol. 284:1141-1151 (1998); Koide et al., “Probing ProteinConformational Changes in Living Cells by Using Designer BindingProteins: Application to the Estrogen Receptor,” Proc. Natl. Acad. Sci.USA 99:1253-1258 (2002), each of which is hereby incorporated byreference in its entirety); and those known as affibodies, which arederived from the stable alpha-helical bacterial receptor domain Z ofstaphylococcal protein A (Nord et al., “Binding Proteins Selected fromCombinatorial Libraries of an alpha-helical Bacterial Receptor Domain,”Nature Biotechnol. 15(8):772-777 (1997), which is hereby incorporated byreference in its entirety).

In preparing these antibody mimics the CDR sequences of the V_(H) and/orV_(L) chains can be grafted into the variable loop regions of theseantibody mimics (see FIGS. 11 and 17 for putative CDR domains). Thegrafting can involve a deletion of at least two amino acid residues upto substantially all but one amino acid residue appearing in aparticular loop region along with the substitution of the CDR sequence.Insertions can be, for example, an insertion of the CDR domain at one ormore locations of a particular loop region. The antibody mimics of thepresent invention preferably possess an amino acid sequence which is atleast 50% homologous to the V_(H) and/or V_(L) chains sequencesdisclosed in the present application. The deletions, insertions, andreplacements on the polypeptides can be achieved using recombinanttechniques beginning with a known nucleotide sequence (see infra).

Methods for monoclonal antibody production may be achieved using thetechniques described herein or other well-known in the art (MONOCLONALANTIBODIES—PRODUCTION, ENGINEERING AND CLINICAL APPLICATIONS (Mary A.Ritter and Heather M. Ladyman eds., 1995), which is hereby incorporatedby reference in its entirety). Generally, the process involves obtainingimmune cells (lymphocytes) from the spleen of a mammal which has beenpreviously immunized with the antigen of interest (i.e., S. aureusglucosaminidase or peptide fragments thereof).

The antibody-secreting lymphocytes are then fused with myeloma cells ortransformed cells, which are capable of replicating indefinitely in cellculture, thereby producing an immortal, immunoglobulin-secreting cellline. Fusion with mammalian myeloma cells or other fusion partnerscapable of replicating indefinitely in cell culture is achieved bystandard and well-known techniques, for example, by using polyethyleneglycol (PEG) or other fusing agents (Milstein and Kohler, “Derivation ofSpecific Antibody-Producing Tissue Culture and Tumor Lines by CellFusion,” Eur. J. Immunol. 6:511 (1976), which is hereby incorporated byreference in its entirety). The immortal cell line, which is preferablymurine, but may also be derived from cells of other mammalian species,is selected to be deficient in enzymes necessary for the utilization ofcertain nutrients, to be capable of rapid growth, and have good fusioncapability. The resulting fused cells, or hybridomas, are cultured, andthe resulting colonies screened for the production of the desiredmonoclonal antibodies. Colonies producing such antibodies are cloned,and grown either in vivo or in vitro to produce large quantities ofantibody.

Thus, a second aspect of present invention relates to a cell line thatexpresses a monoclonal antibody of the present invention. In oneembodiment the monoclonal antibody of the present invention is producedby a hybridoma cell line designated as 1C11, 1E12, 2D11, 3A8, or 3H6. Inanother embodiment, the monoclonal antibody of the present invention (ora binding portion thereof) is produced by a recombinant cell or cellline.

As noted above, monoclonal antibodies can be made using recombinant DNAmethods as described in U.S. Pat. No. 4,816,567 to Cabilly et al., whichis hereby incorporated by reference in its entirety. The polynucleotidesencoding a monoclonal antibody are isolated from mature B-cells orhybridoma cells, for example, by RT-PCR using oligonucleotide primersthat specifically amplify the genes encoding the heavy and light chainsof the antibody. The isolated polynucleotides encoding the heavy andlight chains are then cloned into suitable expression vectors, whichwhen transfected into host cells such as E. coli cells, simian COScells, Chinese hamster ovary (CHO) cells, or myeloma cells that do nototherwise produce immunoglobulin protein, generate host cells thatexpress and secrete monoclonal antibodies. Also, recombinant monoclonalantibodies or fragments thereof of the desired species can be isolatedfrom phage display libraries (McCafferty et al., “Phage Antibodies:Filamentous Phage Displaying Antibody Variable Domains,” Nature348:552-554 (1990); Clackson et al., “Making Antibody Fragments usingPhage Display Libraries,” Nature 352:624-628 (1991); and Marks et al.,“By-Passing Immunization. Human Antibodies from V-Gene LibrariesDisplayed on Phage,” J. Mol. Biol. 222:581-597 (1991), which are herebyincorporated by reference in their entirety).

The present invention also includes a nucleic acid molecule encoding apolypeptide of the present invention. In one embodiment the nucleic acidis DNA. Examples of such DNA sequences are those that comprise a V_(H)and/or V_(L) encoding sequence of the present invention. DNA sequenceencoding for hybridoma 2D11 V_(H) (closest germ line match: J558.18.108)has the nucleotide sequence (SEQ ID NO: 21) as follows:

GAGGTGCAGCTGCAGGAGTCTGGACCTGTGCTGGTGAAGCCTGGGGCTTCAGTGAAGATGTCCTGTAAGGCTTCTGGATACACATTCACTGACTACTATATGAACTGGGTGAAGCAGAGCCATGGAAAGAGCCTTGAGTGGATTGGAGTTATTAATCCTTACAACGGTGATACTACCTACAGCCAGAAGTTCAAGGGCAAGGCCACATTGACTGTTGACAAGTCCTCCAGCACAGCCTACATGGAGCTCAACAGCCTGACATCTGAGGACTCTGCAGTCTATTACTGTGCAAGAAATTACGACGAGTACTTCGATGTCTGGGGCACAGGGACCACGGTCACCGTCTCCTCAGCCAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCNGGTCAAG GGC

DNA sequence encoding for hybridoma 3H6 V_(H) (closest germ line match:J558.18.108) has the nucleotide sequence (SEQ ID NO: 23) as follows:

GAGGTGCAGCTGCAGGAGTCTGGACCTGTGCTGGTGAAGCCTGGGGCTTCAGTGAAGCTGTCCTGTAAGGCTTCTGGATACACATTCACTGACTACTTTATGAACTGGGTGAAGCAGAGCCATGGAAAGAGCCTTGAGTGGATTGGAGTTATTAATCCTTTCAACGGTGGTAATAGGTACAACCAGAACTTCAAGGGCAAGGCCACATTGACTGTTGACAAGTCCTCCAGCACAGCCTACATGGAGCTCAACAGCCTGACATCTGAGGACTCTGCAGTCTATTACTGTGCAAGAGGGGACTATGACTCCCCCTGGTTTGATTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCAGCCAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTGGTCAAGGGCTATTCCCNGAGCCAGTG

DNA sequence encoding for hybridoma 1E12 V_(H) (closest germ line match:7183.46 VH7) has the nucleotide sequence (SEQ ID NO: 25) as follows:

GAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTCGTGAAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTACCTATGTCATGTCTTGGGTTCGCCAGACTCCGGAAAAGAGGCTGGAGTGGGTCGCAACCATTAGTGATGGTGGTGGTCATACTTACTATCTAGACAATGTAAAGGGCCGATTCACCATCTCCAGAGACAATGCCAAGAACAACCTGTACCTGCACATGAGCCATCTGAAGTCTGAGGACACAGCCATGTATTACTGTGCAAGAGCTTACTACGGTAGTAGTTACGACGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAGCCAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTGGTCAAGGGC

DNA sequence encoding for hybridoma 3A8 V_(H) (closest germ line match:VHJ606.4.8.2) has the nucleotide sequence (SEQ ID NO: 27) as follows:

GAGGTGCAGCTGCAGGAGTCTGGAGGAGGCTTGGTGCAACCTGGAGGATCCATGAAACTCTCTTGTGCTGCCTCTGGATTCACTTTTAGTGACGCCTGGATGGACTGGGTCCGCCAGTCTCCAGAGAAGGGGCTTGAGTGGGTTGCTGAAATTAAAGACAAAACTAATAATCATGCAACATACTATGCTGAGTCTGTGAAAGGGAGGTTCACCATCTCAAGAGATGTTTCCAAAAGTCGTGTCTTCCTGCAAATGAACAGCTTAAGACCTGAAGACACTGGCATTTATTACTGTACGTCTGGGCCATATTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCAGCCAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTGGTCAAGGGCTATTTCCCTGAG

DNA sequence encoding for hybridoma 1C11 V_(H) (closest germline match:VH 9-15, DST4-057B1-6, JH3) has the nucleotide sequence (SEQ ID NO: 29)as follows:

CAGATCCAGTTGGTACAGTCTGGACCTGAGCTGAAGAAGCCTGGAGAGACAGTCAAGATCTCCTGCAAGGCTTCTGGGTATACCTTCACAACGTATGGAATGAGCTGGGTGAATCAGGCTCCAGGAAAGGGTTTAAAGTGGATGGGCTGGATAAACACCTACTCTGGAGTGCCAACATATGCTGATGACTTCAAGGGACGGTTTGTCTTCTCTTTGGAAACCTCTGCCAGCACTGCCTATTTGCAGATCAACAACCTCAAAAATGAGGACACGGCTACATATTTCTGTGCAAGAGAGGAGTACAGCTCAGGCTACGCGGCCTGGTTTCCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA

DNA sequence encoding for the 2D11 V_(L) (closest germ line match: at4)has the nucleotide sequence (SEQ ID NO: 22) as follows:

GATATTGTGATGACCCAGTCTCCAGCAATCATGTCTGCATCTCCAGGGGAGAAGGTCACCATGACCTGCAGTGCCAGCTCAAGTGTAAGTTACATGTACTGGTACCAGCAGAAGCCAGGATCCTCCCCCAGACTCCTGATTTATGACACATCCAACCTGGCTTCTGGAGTCCCTGTTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTCTCACAATCAGCCGAATGGAGGCTGAGGATGCTGCCACTTATTACTGCCAGCAGTGGAGTAGTTACCCGCTCACGTTCGGT

DNA sequence encoding for the 3H6 V_(L) (closest germ line match: cp9,JK1) has the nucleotide sequence (SEQ ID NO: 24) as follows:

CAGATGACACAGACTACGTCCTCCCTGTCTGCCTCTCTGGGAGACAGAGTCACCATCAGTTGCAGTGCAAGTCAGGGCATTAGCAATTATTTAAACTGGTATCAGCAGAAACCAGATGGAACTGTTAAACTCCTGATCTATTACACATCAAGTTTACACTCAGGAGTCCCATCAAGGTTCAGTGGCGGTGGGTCTGGGACAGATTATTCTCTCTCCATCAGCAACCTGGAACCTGAAGATATTGCCACTTACTATTGTCAGCAGTATAGTAAGCTTCCTTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA

DNA sequence encoding for the 1E12 V_(L) (closest germ line match: ai4)has the nucleotide sequence (SEQ ID NO: 26) as follows:

GATATTGTGATCACCCAGTCTCCAGCAATCATGTCTGCATCTCTAGGGGAACGGGTCACCATGACCTGCACTGCCAGCTCAAGTGTAAGTTCCAGTTACTTACACTGGTACCAGCAGAAGCCAGGATCCTCCCCCAAACTNTGGATTTATAGCACATCCAACCTGGCTTCTGGAGTCCCAGCTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTCTCACAATCAGCAGCATGGAGGCTGAAGATGCTGCCACTTATTACTGCCACCAGTATCATCGTTCCCCATGGACGTTCGGTGGAGGCACC

DNA sequence encoding for the 3A8 V_(L) (closest germ line match: KV19-25, JK2) has the nucleotide sequence (SEQ ID NO: 28) as follows:

GACATTGTGATGACCCAGTCTCACAAATTCATGTCCACATCAGTAGGAGACAGGGTCAGCATCACCTGCAAGGCCAGTCAGGACGTGAGTACTGCTGTAGCCTGGTATCAACAAAAACCAGGGCAATCTCCTAAACTACTGATTTACTGGACATCCACCCGGCACACTGGAGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTTACTCTCACCATCAGCAGTGTGCAGGCTAAAGACCTGGCACTTTATTACTGTCAGCAACATTATACCACTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAA ATAAAA

DNA sequence encoding for 1C11 V_(L) (closest germ line match: VK23-43,JK5) has the nucleotide sequence (SEQ ID NO: 30) as follows:

GATATTGTGCTAACTCAGTCTCCAGCCACCCTGTCTGTGACTCCAGGAGATAGCGTCAGTCTTTCCTGCAGGGCCAGCCAAAGTATTAGCAACAACCTACACTGGTATCAACAAAAATCACATGAGTCTCCAAGGCTTCTCATCGAATATGCTTCCCGGTCCATCTCTGGGATCCCCTCTAGGTTCAGTGGCGGTGGATCAGGGACAGATTTCACTCTCAGTATCAACAGTGTGGAGTCTGAAGATTTTGGATTGTATTTCTGTCAACAGAGTAACAGCTGGCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAG CTGAAA

Still a further aspect of the present invention is a DNA constructcomprising a DNA molecule that encodes an antibody or binding portion ofthe present invention, a promoter-effective DNA molecule operablycoupled 5′ of the DNA molecule, and a transcription termination DNAmolecule operably coupled 3′ of the DNA molecule. The present inventionalso encompasses an expression vector into which the DNA construct ofthe present invention is inserted. A synthetic gene for the polypeptidesof the present invention can be designed such that it includesconvenient restriction sites for ease of mutagenesis and uses specificcodons for high-level protein expression (Gribskov et al., “The CodonPreference Plot: Graphic Analysis of Protein Coding Sequences andPrediction of Gene Expression,” Nuc. Acids. Res. 12:539-549 (1984),which is hereby incorporated by reference in its entirety).

The gene may be assembled as follows: first the gene sequence can bedivided into parts with boundaries at designed restriction sites; foreach part, a pair of oligonucleotides that code opposite strands andhave complementary overlaps of about 15 bases can be synthesized; thetwo oligonucleotides can be annealed and single strand regions can befilled in using the Klenow fragment of DNA polymerase; thedouble-stranded oligonucleotide can be cloned into a vector, such as,the pET3a vector (Novagen) using restriction enzyme sites at the terminiof the fragment and its sequence can be confirmed by a DNA sequencer;and these steps can be repeated for each of the parts to obtain thewhole gene. This approach takes more time to assemble a gene than theone-step polymerase chain reaction (PCR) method (Sandhu et al., “DualAsymetric PCR: One-Step Construction of Synthetic Genes,” BioTech.12:14-16 (1992), which is hereby incorporated by reference in itsentirety). Mutations could likely be introduced by the low fidelityreplication by Taq polymerase and would require time-consuminggene-editing. Recombinant DNA manipulations can be performed accordingto SAMBROOK & RUSSELL, MOLECULAR CLONING: A LABORATORY MANUAL (2d ed.1989), which is hereby incorporated by reference in its entirety, unlessotherwise stated. To avoid the introduction of mutations during one-stepPCR, high fidelity/low error polymerases can be employed as is known inthe art.

Desired mutations can be introduced to the polypeptides sequence of thepresent invention using either cassette mutagenesis, oligonucleotidesite-directed mutagenesis techniques (Deng & Nickoloff, “Site-DirectedMutagenesis of Virtually any Plasmid by Eliminating a Unique Site,”Anal. Biochem. 200:81-88 (1992), which is hereby incorporated byreference in its entirety), or Kunkel mutagenesis (Kunkel et al., “Rapidand Efficient Site-Specific Mutagenesis Without Phenotypic Selection,”Proc. Natl. Acad. Sci. USA 82:488-492 (1985); Kunkel et al., “Rapid andEfficient Site-Specific Mutagenesis Without Phenotypic Selection,”Methods Enzymol. 154:367-382 (1987), which are hereby incorporated byreference in their entirety).

Both cassette mutagenesis and site-directed mutagenesis can be used toprepare specifically desired nucleotide coding sequences. Cassettemutagenesis can be performed using the same protocol for geneconstruction described above and the double-stranded DNA fragment codinga new sequence can be cloned into a suitable expression vector. Manymutations can be made by combining a newly synthesized strand (codingmutations) and an oligonucleotide used for the gene synthesis.Regardless of the approach utilized to introduce mutations into thenucleotide sequence encoding a polypeptide according to the presentinvention, sequencing can be performed to confirm that the designedmutations (and no other mutations) were introduced by mutagenesisreactions.

In contrast, Kunkel mutagenesis can be utilized to randomly produce aplurality of mutated polypeptide coding sequences which can be used toprepare a combinatorial library of polypeptides for screening.Basically, targeted loop regions (or C-terminal or N-terminal tailregions) can be randomized using the NNK codon (N denoting a mixture ofA, T, G, C, and K denoting a mixture of G and T) (Kunkel et al., “Rapidand Efficient Site-Specific Mutagenesis Without Phenotypic Selection,”Methods Enzymol. 154:367-382 (1987), which is hereby incorporated byreference in its entirety).

Regardless of the approach used to prepare the nucleic acid moleculesencoding the polypeptide according to the present invention, the nucleicacid can be incorporated into host cells using conventional recombinantDNA technology. Generally, this involves inserting the DNA molecule intoan expression system to which the DNA molecule is heterologous (i.e.,not normally present). The heterologous DNA molecule is inserted intothe expression system or vector in sense orientation and correct readingframe. The vector contains the necessary elements (promoters,suppressers, operators, transcription termination sequences, etc.) forthe transcription and translation of the inserted protein-codingsequences. A recombinant gene or DNA construct can be prepared prior toits insertion into an expression vector. For example, using conventionalrecombinant DNA techniques, a promoter-effective DNA molecule can beoperably coupled 5′ of a DNA molecule encoding the polypeptide and atranscription termination (i.e., polyadenylation sequence) can beoperably coupled 3′ thereof.

In accordance with this aspect of the invention, the polynucleotides ofthe present invention are inserted into an expression system or vectorto which the molecule is heterologous. The heterologous nucleic acidmolecule is inserted into the expression system or vector in propersense (5′→3′) orientation relative to the promoter and any other 5′regulatory molecules, and correct reading frame. The preparation of thenucleic acid constructs can be carried out using standard cloningmethods well known in the art as described by SAMBROOK & RUSSELL,MOLECULAR CLONING: A LABORATORY MANUAL (Cold Springs Laboratory Press,2001), which is hereby incorporated by reference in its entirety. U.S.Pat. No. 4,237,224 to Cohen and Boyer, which is hereby incorporated byreference in its entirety, also describes the production of expressionsystems in the form of recombinant plasmids using restriction enzymecleavage and ligation with DNA ligase.

Suitable expression vectors include those which contain replicon andcontrol sequences that are derived from species compatible with the hostcell. For example, if E. coli is used as a host cell, plasmids such aspUC19, pUC18 or pBR322 may be used. When using insect host cells,appropriate transfer vectors compatible with insect host cells include,pVL1392, pVL1393, pAcGP67 and pAcSecG2T, which incorporate a secretorysignal fused to the desired protein, and pAcGHLT and pAcHLT, whichcontain GST and 6xHis tags (BD Biosciences, Franklin Lakes, N.J.). Viralvectors suitable for use in carrying out this aspect of the inventioninclude, adenoviral vectors, adeno-associated viral vectors, vacciniaviral vectors, nodaviral vectors, and retroviral vectors. Other suitableexpression vectors are described in SAMBROOK AND RUSSELL, MOLECULARCLONING: A LABORATORY MANUAL (Cold Springs Laboratory Press, 2001),which is hereby incorporated by reference in its entirety. Many knowntechniques and protocols for manipulation of nucleic acids, for examplein preparation of nucleic acid constructs, mutagenesis, sequencing,introduction of DNA into cells and gene expression, and analysis ofproteins, are described in detail in CURRENT PROTOCOLS IN MOLECULARBIOLOGY (Fred M. Ausubel et al. eds., 2003), which is herebyincorporated by reference in its entirety.

Different genetic signals and processing events control many levels ofgene expression (e.g., DNA transcription and messenger RNA (“mRNA”)translation) and subsequently the amount of antibodies or antibodyfragments that are produced and expressed by the host cell.Transcription of DNA is dependent upon the presence of a promoter, whichis a DNA sequence that directs the binding of RNA polymerase, andthereby promotes mRNA synthesis. Promoters vary in their “strength”(i.e., their ability to promote transcription). For the purposes ofexpressing a cloned gene, it is desirable to use strong promoters toobtain a high level of transcription and, hence, expression. Dependingupon the host system utilized, any one of a number of suitable promotersmay be used. For instance, when using E. coli, its bacteriophages, orplasmids, promoters such as the T7 phage promoter, lac promoter, trppromoter, recA promoter, ribosomal RNA promoter, the P_(R) and P_(L)promoters of coliphage lambda and others, including but not limited, tolacUV5, ompF, bla, lpp, and the like, may be used to direct high levelsof transcription of adjacent DNA segments. Additionally, a hybridtrp-lacUV5 (tac) promoter or other E. coli promoters produced byrecombinant DNA or other synthetic DNA techniques may be used to providefor transcription of the inserted gene. When using insect cells,suitable baculovirus promoters include late promoters, such as 39Kprotein promoter or basic protein promoter, and very late promoters,such as the p10 and polyhedron promoters. In some cases it may bedesirable to use transfer vectors containing multiple baculoviralpromoters. Common promoters suitable for directing expression inmammalian cells include, without limitation, SV40, MMTV,metallothionein-1, adenovirus Ela, CMV, immediate early, immunoglobulinheavy chain promoter and enhancer, and RSV-LTR. The promoters can beconstitutive or, alternatively, tissue-specific or inducible. Inaddition, in some circumstances inducible (TetOn) promoters can be used.

Translation of mRNA in prokaryotes depends upon the presence of theproper prokaryotic signals, which differ from those of eukaryotes.Efficient translation of mRNA in prokaryotes requires a ribosome bindingsite called the Shine-Dalgarno (“SD”) sequence on the mRNA. Thissequence is a short nucleotide sequence of mRNA that is located beforethe start codon, usually AUG, which encodes the amino-terminalmethionine of the protein. The SD sequences are complementary to the3′-end of the 16S rRNA (ribosomal RNA) and promote binding of mRNA toribosomes by duplexing with the rRNA to allow correct positioning of theribosome. For a review on maximizing gene expression, see Roberts andLauer, “Maximizing Gene Expression on a Plasmid Using Recombination InVitro,” Methods in Enzymology, 68:473-82 (1979), which is herebyincorporated by reference in its entirety.

The present invention also includes a host cell transformed with the DNAconstruct of the present invention. The host cell can be a prokaryote ora eukaryote. Host cells suitable for expressing the polypeptides of thepresent invention include any one of the more commonly available gramnegative bacteria. Suitable microorganisms include Pseudomonasaeruginosa, Escherichia coli, Salmonella gastroenteritis (typhimirium),S. typhi, S. enteriditis, Shigella flexneri, S. sonnie, S. dysenteriae,Neisseria gonorrhoeae, N. meningitides, Haemophilus influenzae, H.pleuropneumoniae, Pasteurella haemolytica, P. multilocida, Legionellapneumophila, Treponema pallidum, T. denticola, T. orales, Borreliaburgdorferi, Borrelia spp., Leptospira interrogans, Klebsiellapneumoniae, Proteus vulgaris, P. morganii, P. mirabilis, Rickettsiaprowazeki, R. typhi, R. richettsii, Porphyromonas (Bacteriodes)gingivalis, Chlamydia psittaci, C. pneumoniae, C. trachomatis,Campylobacter jejuni, C. intermedis, C. fetus, Helicobacter pylori,Francisella tularenisis, Vibrio cholerae, Vibrio parahaemolyticus,Bordetella pertussis, Burkholderie pseudomallei, Brucella abortus, B.susi, B. melitensis, B. canis, Spirillum minus, Pseudomonas mallei,Aeromonas hydrophila, A. salmonicida, and Yersinia pestis.

In addition to bacteria cells, animal cells, in particular mammalian andinsect cells, yeast cells, fungal cells, plant cells, or algal cells arealso suitable host cells for transfection/transformation of therecombinant expression vector carrying an isolated polynucleotidemolecule of the present invention. Mammalian cell lines commonly used inthe art include Chinese hamster ovary cells, HeLa cells, baby hamsterkidney cells, COS cells, and many others. Suitable insect cell linesinclude those susceptible to baculoviral infection, including Sf9 andSf21 cells.

Methods for transforming/transfecting host cells with expression vectorsare well-known in the art and depend on the host system selected, asdescribed in SAMBROOK & RUSSELL, MOLECULAR CLONING: A LABORATORY MANUAL(Cold Springs Laboratory Press, 2001), which is hereby incorporated byreference in its entirety. For bacterial cells, suitable techniquesinclude calcium chloride transformation, electroporation, andtransfection using bacteriophage. For eukaryotic cells, suitabletechniques include calcium phosphate transfection, DEAE-Dextran,electroporation, liposome-mediated transfection, and transduction usingretrovirus or any other viral vector. For insect cells, the transfervector containing the polynucleotide construct of the present inventionis co-transfected with baculovirus DNA, such as AcNPV, to facilitate theproduction of a recombinant virus. Subsequent recombinant viralinfection of Sf cells results in a high rate of recombinant proteinproduction. Regardless of the expression system and host cell used tofacilitate protein production, the expressed antibodies, antibodyfragments, or antibody mimics of the present invention can be readilypurified using standard purification methods known in the art anddescribed in PHILIP L. R. BONNER, PROTEIN PURIFICATION (Routledge 2007),which is hereby incorporated by reference in its entirety.

The polynucleotide(s) encoding a monoclonal antibody can further bemodified using recombinant DNA technology to generate alternativeantibodies. For example, the constant domains of the light and heavychains of a mouse monoclonal antibody can be substituted for thoseregions of a human antibody to generate a humanized (or chimeric)antibody, as discussed above. Alternatively, the constant domains of thelight and heavy chains of a mouse monoclonal antibody can be substitutedfor a non-immunoglobulin polypeptide to generate a fusion antibody. Inother embodiments, the constant regions are truncated or removed togenerate the desired antibody fragment of a monoclonal antibody.Furthermore, site-directed or high-density mutagenesis of the variableregion can be used to optimize specificity and affinity of a monoclonalantibody.

A third aspect of the present invention is related to a pharmaceuticalcomposition comprising a carrier and one or more monoclonal antibodiesor one or more binding portions thereof in accordance with the presentinvention. This pharmaceutical composition may contain two or moreantibodies or binding fragments where all antibodies or bindingfragments recognize the same epitope. Alternatively, the pharmaceuticalcomposition may contain an antibody or binding fragment mixture whereone or more antibodies or binding fragments recognize one epitope of S.aureus Gmd and one or more antibodies or binding fragments recognize adifferent epitope of S. aureus Gmd. For example, the mixture may containone or more antibodies of the present invention that bind specificallyto an R3 domain of Staphylococcus aureus glucosaminidase in combinationwith any other antibody that binds to glucosaminidase, such as anantibody that binds to the catalytic domain of glucosaminidase. Thepharmaceutical composition of the present invention further contains apharmaceutically acceptable carrier or other pharmaceutically acceptablecomponents as described infra

In accordance with one embodiment, the pharmaceutical compositionincludes one or more of mAbs 1C11, 2D11, 3H6, 1E12, and 3A8 in apharmaceutically acceptable carrier. In accordance with anotherembodiment, the pharmaceutical composition includes two or more of mAbs1C11, 2D11, 3H6, 1E12, and 3A8 in a pharmaceutically acceptable carrier.

A pharmaceutical composition containing the monoclonal antibodies of thepresent invention can be administered to a subject having or at risk ofhaving Staphylococcus infection. Various delivery systems are known andcan be used to administer the antibodies of the present invention.Methods of introduction include but are not limited to intradermal,intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal,epidural, and oral routes. The therapeutic agent can be administered,for example by infusion or bolus injection, by absorption throughepithelial or mucocutaneous linings (e.g., oral mucosa, rectal andintestinal mucosa, and the like) and can be administered together withother biologically active agents, such as chemotherapeutic agents,antibiotic agents, or other immunotherapeutic agents. Administration canbe systemic or local, i.e., at a site of Staph infection or directly toa surgical or implant site.

The pharmaceutical composition of the present invention can furthercomprise administering a second therapeutic agent to the patient,wherein the second therapeutic agent is an antibiotic agent orimmunotherapeutic agent. Exemplary antibiotic agents include, withoutlimitation, vancomycin, tobramycin, cefazolin, erythromycin,clindamycin, rifampin, gentamycin, fusidic acid, minocycline,co-trimoxazole, clindamycin, linezolid, quinupristin-dalfopristin,daptomycin, tigecycline, dalbavancin, telavancin, oritavancin,ceftobiprole, ceftaroline, iclaprim, the carbapenem CS-023/RO-4908463,and combinations thereof. Exemplary immunotherapeutic agents include,without limitation, tefibazumab, BSYX-A110, Aurexis™, and combinationsthereof. The above lists of antibiotic agents and immunotherapeuticagents are intended to be non-limiting examples; thus, other antibioticagents or immunotherapeutic agents are also contemplated. Combinationsor mixtures of the second therapeutic agent can also be used for thepurposes of the present invention. These agents can be administeredcontemporaneously or as a single formulation.

The pharmaceutical composition typically includes one or morepharmaceutical carriers (e.g., sterile liquids, such as water and oils,including those of petroleum, animal, vegetable or synthetic origin,such as peanut oil, soybean oil, mineral oil, sesame oil and the like).Water is a more typical carrier when the pharmaceutical composition isadministered intravenously. Saline solutions and aqueous dextrose andglycerol solutions can also be employed as liquid carriers, particularlyfor injectable solutions. Suitable pharmaceutical excipients include,for example, starch, glucose, lactose, sucrose, gelatin, malt, rice,flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc,sodium chloride, dried skim milk, glycerol, propylene glycol, water,ethanol, and the like. The composition, if desired, can also containminor amounts of wetting or emulsifying agents, or pH buffering agents.These compositions can take the form of solutions, suspensions,emulsion, tablets, pills, capsules, powders, sustained-releaseformulations and the like. The composition can be formulated as asuppository, with traditional binders and carriers such astriglycerides. Oral formulations can include standard carriers such aspharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate, etc. Examples ofsuitable pharmaceutical carriers are described in “Remington'sPharmaceutical Sciences” by E. W. Martin. Such compositions will containa therapeutically effective amount of the nucleic acid or protein,typically in purified form, together with a suitable amount of carrierso as to provide the form for proper administration to the patient. Theformulations correspond to the mode of administration.

Effective doses of the compositions of the present invention, for thetreatment of the above described bacterial infections vary dependingupon many different factors, including mode of administration, targetsite, physiological state of the patient, other medicationsadministered, and whether treatment is prophylactic or therapeutic. Inprophylactic applications, a relatively low dosage is administered atrelatively infrequent intervals over a long period of time. Somepatients continue to receive treatment for the rest of their lives. Intherapeutic applications, a relatively high dosage at relatively shortintervals is sometimes required until progression of the disease isreduced or terminated, and preferably until the patient shows partial orcomplete amelioration of symptoms of disease. Thereafter, the patientcan be administered a prophylactic regime. For prophylactic treatmentagainst Staphylococcus bacterial infection, it is intended that thepharmaceutical composition(s) of the present invention can beadministered prior to exposure of an individual to the bacteria and thatthe resulting immune response can inhibit or reduce the severity of thebacterial infection such that the bacteria can be eliminated from theindividual. For example, the monoclonal antibody or the pharmaceuticalcomposition can be administered prior to, during, and/or immediatelyfollowing a surgical procedure, such as joint replacement or any surgeryinvolving a prosthetic implant.

For passive immunization with an antibody or binding fragment of thepresent invention, the dosage ranges from about 0.0001 to about 100mg/kg, and more usually about 0.01 to about 5 mg/kg, of the host bodyweight. For example, dosages can be about 1 mg/kg body weight or about10 mg/kg body weight, or within the range of about 1 to about 10 mg/kg.An exemplary treatment regime entails administration once per every twoweeks or once a month or once every 3 to 6 months. In some methods, twoor more monoclonal antibodies with different binding specificities areadministered simultaneously, in which case the dosage of each antibodyadministered falls within the ranges indicated. Antibody is usuallyadministered on multiple occasions. Intervals between single dosages canbe daily, weekly, monthly, or yearly. In some methods, dosage isadjusted to achieve a plasma antibody concentration of 1-1000 μg/ml andin some methods 25-300 μg/ml. Alternatively, antibody can beadministered as a sustained release formulation, in which case lessfrequent administration is required. Dosage and frequency vary dependingon the half-life of the antibody in the patient. In general, humanantibodies show the longest half life, followed by humanized antibodies,chimeric antibodies, and nonhuman antibodies.

A further aspect of the invention relates to an active vaccine (e.g.,pharmaceutical composition) that includes a carrier and an antigenicmolecule comprising at least a fragment of the S. aureusglucosaminidase. The antigenic molecule can be in the form of (i) afusion protein that includes the glucosaminidase polypeptide and anadjuvant polypeptide or (ii) an immunogenic conjugate that includes theglucosaminidase polypeptide conjugated to another immunogenic molecule.

By way of example, and without limitation, suitable fusion proteins ofthe present invention include those containing an adjuvant polypeptideselected from the group of flagellin, human papillomavirus (HPV) L1 orL2 proteins, herpes simplex glycoprotein D (gD), complement C4 bindingprotein, a toll-like receptor-4 (TLR4) ligand, and IL-1β.

The fusion polypeptide or protein of the present invention can begenerated using standard techniques known in the art. For example, thefusion polypeptide can be prepared by translation of an in-frame fusionof the polynucleotide sequences of the present invention and theadjuvant, i.e., a hybrid or chimeric gene. The hybrid gene encoding thefusion polypeptide is inserted into an expression vector which is usedto transform or transfect a host cell. Alternatively, the polynucleotidesequence encoding the polypeptide of the present invention is insertedinto an expression vector in which the polynucleotide encoding theadjuvant is already present. The peptide adjuvant of the fusion proteincan be fused to the N-, or preferably, to the C-terminal end of theglucosaminidase polypeptide of the present invention.

Fusions between the polypeptides of the present invention and theprotein adjuvant may be such that the amino acid sequence of thepolypeptide of the present invention is directly contiguous with theamino acid sequence of the adjuvant. Alternatively, the polypeptideportion may be coupled to the adjuvant by way of a short linkersequence. Suitable linker sequences include glycine rich linkers (e.g.,GGGS₂₋₃), serine-rich linkers (e.g., GS_(N)), or other flexibleimmunoglobulin linkers as disclosed in U.S. Pat. No. 5,516,637 to Huanget al, which is hereby incorporated by reference in its entirety.

Suitable immunogenic conjugates of the present invention include, butare not limited to, those containing an immunogenic carrier moleculecovalently or non-covalently bonded to any glucosaminidase polypeptide.Any suitable immunogenic carrier molecule can be used. Exemplaryimmunogenic carrier molecules include, but are in no way limited to,bovine serum albumin, chicken egg ovalbumin, keyhole limpet hemocyanin,tetanus toxoid, diphtheria toxoid, thyroglobulin, a pneumococcalcapsular polysaccharide, CRM 197, and a meningococcal outer membraneprotein.

The pharmaceutical composition in the form of an active vaccine can alsoinclude an effective amount of a separate adjuvant. Suitable adjuvantsfor use in the present invention include, without limitation, aluminumhydroxide, aluminum phosphate, aluminum potassium sulfate, berylliumsulfate, silica, kaolin, carbon, water-in-oil emulsions, oil-in-wateremulsions, muramyl dipeptide, bacterial endotoxin, lipid, Quil A, and/ornon-infective Bordetella pertussis.

The choice of an adjuvant depends on the stability of the immunogenicformulation containing the adjuvant, the route of administration, thedosing schedule, the efficacy of the adjuvant for the species beingvaccinated, and, in humans, a pharmaceutically acceptable adjuvant isone that has been approved or is approvable for human administration bypertinent regulatory bodies. For example, alum, MPL or IncompleteFreund's adjuvant (Chang et al., Advanced Drug Delivery Reviews32:173-186 (1998), which is hereby incorporated by reference in itsentirety) alone or optionally all combinations thereof are suitable forhuman administration.

In prophylactic applications, pharmaceutical compositions containing theimmunogenic glucosaminidase polypeptides are administered to a patientsusceptible to, or otherwise at risk of, the bacterial infection in anamount sufficient to eliminate or reduce the risk, lessen the severity,or delay the outset of the disease, including biochemical, histologicand/or behavioral symptoms of the disease, its complications andintermediate pathological phenotypes presented during development of thedisease. In therapeutic applications, pharmaceutical compositionscontaining a monoclonal antibody or binding fragment according to thepresent invention are administered to a patient suspected of, or alreadysuffering from, such a disease in an amount sufficient to cure, or atleast partially arrest, the symptoms of the disease (biochemical,histologic and/or behavioral), including its complications andintermediate pathological phenotypes in development of the disease. Anamount adequate to accomplish therapeutic or prophylactic treatment isdefined as a therapeutically- or prophylactically-effective dose, whichis identified supra. In both prophylactic and therapeutic regimes,agents are usually administered in several dosages until a sufficientresponse has been achieved. Typically, the response is monitored andrepeated dosages are given if the response starts to wane.

Treatment dosages should be titrated to optimize safety and efficacy.The amount of immunogen depends on whether adjuvant is alsoadministered, with higher dosages being required in the absence ofadjuvant. The amount of an immunogen for administration sometimes variesfrom 1-500 μg per patient and more usually from 5-500 μg per injectionfor human administration. Occasionally, a higher dose of 1-2 mg perinjection is used. Typically about 10, 20, 50, or 100 μg is used foreach human injection. The mass of immunogen also depends on the massratio of immunogenic epitope within the immunogen to the mass ofimmunogen as a whole. Typically, 10⁻³ to 10⁻⁵ micromoles of immunogenicepitope are used for each microgram of immunogen. The timing ofinjections can vary significantly from once a day, to once a year, toonce a decade. On any given day that a dosage of immunogen is given, thedosage is greater than 1 μg/patient and usually greater than 10μg/patient if adjuvant is also administered, and greater than 10μg/patient and usually greater than 100 μg/patient in the absence ofadjuvant. A typical regimen consists of an immunization followed bybooster injections at time intervals, such as 6 week intervals. Anotherregimen consists of an immunization followed by booster injections 1, 2,and 12 months later. Alternatively, booster injections can be providedon a regular or irregular basis, as indicated by monitoring of immuneresponse.

A fourth aspect the present invention relates to a method of treating anS. aureus infection that includes administering to a patient having anS. aureus infection an effective amount of a monoclonal antibody orbinding fragment thereof or a pharmaceutical composition of the presentinvention.

In one embodiment of this aspect of the invention the method of treatingS. aureus infection further comprises repeating said administering. Themethod of treating S. aureus infection can be such that theadministering is carried out systemically or carried out directly to asite of the S. aureus infection.

The method of treating S. aureus infection can be used to treat S.aureus infection at sites which include, without limitation, infectionof the skin, muscle, cardiac, respiratory tract, gastrointestinal tract,eye, kidney and urinary tract, and bone or joint infections.

In one embodiment, this method is carried out to treat osteomyelitis byadministering an effective amount of the monoclonal antibody or bindingfragment thereof or the pharmaceutical composition of the presentinvention to a patient having an S. aureus bone or joint infection.Administration of these agents or compositions can be carried out usingany of the routes described supra; however, administration directly tothe site of the bone or joint infection is preferred.

A sixth aspect of the present invention relates to a method ofintroducing an orthopedic implant into a patient that includesadministering to a patient in need of an orthopedic implant an effectiveamount of a monoclonal antibody, binding portion, or pharmaceuticalcomposition of the present invention, and introducing the orthopedicimplant into the patient.

In one embodiment, the method of introducing an orthopedic implantincludes administering to the patient in need of the orthopedic implantan effective amount of a monoclonal antibody or binding fragment or apharmaceutical composition containing the same, directly to the site ofimplantation. Alternatively, or in addition, the orthopedic implant canbe coated or treated with the monoclonal antibody or binding fragment ora pharmaceutical composition containing the same before, during, orimmediately after implantation thereof at the implant site.

The orthopedic implant can be a joint prosthesis, graft or syntheticimplant. Exemplary joint prosthetics includes, without limitation, aknee prosthetic, hip prosthetic, finger prosthetic, elbow prosthetic,shoulder prosthetic, temperomandibular prosthetic, and ankle prosthetic.Other prosthetics can also be used. Exemplary grafts or syntheticimplants include, without limitation, an artificial intervertebral disk,meniscal implant, or a synthetic or allograft anterior cruciateligament, medial collateral ligament, lateral collateral ligament,posterior cruciate ligament, Achilles tendon, and rotator cuff Othergrafts or implants can also be used.

In one embodiment, the method of introducing an orthopedic implant isintended to encompass the process of installing a revision total jointreplacement. Where infection, particularly Staph infection of anoriginal joint replacement occurs, the only viable treatment is arevision total joint replacement. In this embodiment, the infected jointprosthesis is first removed and then the patient is treated for theunderlying infection. Treatment of the infection occurs over an extendedperiod of time (i.e. 6 months), during which time the patient isimmobile (or has only limited mobility) and receives high doses ofantibiotics to treat the underlying infection and optionally one or moremonoclonal antibodies or binding portions, or pharmaceuticalcompositions of the present invention. Upon treatment of the underlyinginfection, the new joint prosthesis is installed. Immediately prior(i.e., within the two weeks preceding new joint prosthesis installation)and optionally subsequent to installation of the new joint prosthesis,the patient is administered one or more monoclonal antibodies or bindingportions, or pharmaceutical compositions of the present invention. Thistreatment can be repeated one or more times during the post-installationperiod. Antibiotic treatment may be administered in combination with orconcurrently with the one or more monoclonal antibodies or bindingportions, or pharmaceutical compositions of the present invention. Thesetreatments are effective to prevent infection or reinfection during therevision total joint replacement.

The methods of treatment according to the present invention can be usedto treat any patient in need, however, the methods are particularlyuseful for immuno-compromised patients of any age, as well as patientsthat are older than 50 years of age.

EXAMPLES

The present invention is illustrated by reference to the followingexamples. These examples are not intended to limit the claimedinvention.

Example 1 A Murine Transtibial Model of Implant-Associated Osteomyelitis

Orthopedic implant-associated OM occurs for both intramedullary devices(i.e. joint prostheses) and transcortical implants (i.e. externalfixation devices, FIG. 1A). Although the infection rate of fixationdevices is 2.5 times greater, and has an incidence of over 8-times thatof total joint prostheses, it is not considered to be as serious becausethe revision surgery is much simpler (Darouiche, “Treatment ofInfections Associated With Surgical Implants,” N. Engl. J. Med.350(14):1422-9 (2004), which is hereby incorporated by reference in itsentirety). While most cases involving an infected transcortical implantcan be resolved in a single surgery to relocate the pin and treating theabscess independently, the majority of infected prostheses must undergotwo revision surgeries (Darouiche, “Treatment of Infections AssociatedWith Surgical Implants,” N. Engl. J. Med. 350(14):1422-9 (2004), whichis hereby incorporated by reference in its entirety). The first isneeded to cure the infection, and the second replaces the prosthesis.Thus, from a clinical significance standpoint, the field has focusedprimarily on models of implant-associated OM that involve anintramedullary device with the UAMS-1 (ATCC 49230) strain of S. aureus(Daum et al., “A Model of Staphylococcus aureus Bacteremia, SepticArthritis, and Osteomyelitis in Chickens,” J. Orthop. Res. 8(6):804-13(1990); Rissing et al., “Model of Experimental Chronic Osteomyelitis inRats,” Infect. Immun. 47(3):581-6 (1985); Passl et al., “A Model ofExperimental Post-Traumatic Osteomyelitis in Guinea Pigs,” J. Trauma24(4):323-6 (1984); Worlock et al., “An Experimental Model ofPost-Traumatic Osteomyelitis in Rabbits,” Br. J. Exp. Pathol.69(2):235-44 (1988); Varshney et al., “Experimental Model ofStaphylococcal Osteomyelitis in Dogs,” Indian J. Exp. Biol. 27(9):816-9(1989); Kaarsemaker et al., “New Model for Chronic Osteomyelitis WithStaphylococcus aureus in Sheep,” Clin. Orthop. Relat. Res. 339:246-52(1997), which are hereby incorporated by reference in their entirety).Unfortunately, this approach has significant limitations, most notablythe inability to generate reproducible (temporal and spatial) lesions.In an effort to overcome this the location of the infection was guidedto the diaphysis by fracturing the tibia immediately after inserting anintramedullary pin containing1×10⁶ CFU, using an Einhorn device asdescribed previously (Zhang et al., “Cyclooxygenase-2 RegulatesMesenchymal Cell Differentiation Into the Osteoblast Lineage and isCritically Involved in Bone Repair,” J. Clin. Invest. 109(11):1405-15(2002), which is hereby incorporated by reference in its entirety). Itwas found that implantation of an infected transcortical pin alwaysproduces lesions adjacent to the pin, and never results in chronic OM inother regions of the tibia or hematogenous spreading in mice (FIGS.1A-C).

To quantify the osteolysis, a time-course study was performed in whichthe infected tibiae were analyzed by μCT (FIGS. 1B-C). These results areconsistent with sequestrum formation in which osteoclastic boneresorption around the infected implant occurs with concomitant reactiveperiosteal bone formation.

The presence of OM in the mice was confirmed histologically. FIGS. 2A-Hdemonstrate that the tibial transcortical pin model contains all of thesalient features of chronic OM including: sequestrum and involucrumformation, osteoclastic resorption of the cortical bone and Gram stainedextracellular bacteria and biofilm that reside in the necrotic bonesurrounding the implant. None of the negative controls, including heatkilled S. aureus and non-pathogenic E. coli, demonstrated thesefeatures.

Example 2 Real Time PCR Quantitation of Osteomyelitis

There are no known methods to quantify OM. Since it is impossible toeffectively extract live bacteria from infected bone to determinebacterial load by classical colony forming units (CFU), a real time PCRmethod was developed to quantify the number of recoverable nuc genes inDNA samples, as is done to test for contamination in cheese (Hein etal., “Comparison of Different Approaches to Quantify Staphylococcusaureus Cells by Real-Time Quantitative PCR and Application of ThisTechnique for Examination of Cheese,” Appl. Environ. Microbiol.67(7):3122-6 (2001), which is hereby incorporated by reference in itsentirety) and blood (Palomares et al., “Rapid Detection andIdentification of Staphylococcus aureus From Blood Culture SpecimensUsing Real-Time Fluorescence PCR,” Diagn. Microbiol. Infect. Dis.45(3):183-9 (2003), which is hereby incorporated by reference in itsentirety), as a surrogate outcome measure of bacterial load.

RTQ-PCR for the S. aureus-specific nuc gene can be performed usingprimers 5′-GCGATTGATGGTGATACGGTT-3′ (SEQ ID NO: 15) and5′-AGCCAAGCCTTGACGAACTAA-3′ (SEQ ID NO: 16) that amplify a previouslydescribed 269-bp product (Hein et al., “Comparison of DifferentApproaches to Quantify Staphylococcus aureus Cells by Real-TimeQuantitative PCR and Application of This Technique for Examination ofCheese,” Appl. Environ. Microbiol. 67(7):3122-6 (2001), which is herebyincorporated by reference in its entirety). The reactions can be carriedout in a final volume of 20 μl consisting of 0.3 μM primers, 1× SybrGreen PCR Super Mix (BioRad, Hercules, Calif.), and 2 μl of the purifiedtibia DNA template. The samples can be assayed using a Rotor-Gene RG3000 (Corbett Research, Sydney, AU).

To control for the integrity of the DNA template between samples,RTQ-PCR can also be performed for the mouse β-actin gene that detects a124-bp product using primers 5′-AGATGTGAATCAGCAAGCAG-3′ (SEQ ID NO: 17)and 5′-GCGCAAGTTAGGTTTTGTCA-3′ (SEQ ID NO: 18). Using PCR primersspecific for murine β-actin, S. aureus nuc, and rRNA genomic DNA, thespecificity of these PCRs and the ability to amplify the predictedproducts was demonstrated (Li et al., “Quantitative Mouse Model ofImplant-Associated Osteomyelitis and the Kinetics of Microbial Growth,Osteolysis, and Humoral Immunity,” J. Orthop. Res. 26(1):96-105 (2008),which is hereby incorporated by reference in its entirety). Then, usingpurified plasmid DNA containing the nuc gene, or S. aureus genomic DNA,a dose response experiment was performed and it was determined that thedetection limit for this RTQ-PCR is ˜100 copies (Li et al.,“Quantitative Mouse Model of Implant-Associated Osteomyelitis and theKinetics of Microbial Growth, Osteolysis, and Humoral Immunity,” J.Orthop. Res. 26(1):96-105 (2008), which is hereby incorporated byreference in its entirety). This assay has been used to quantify the invivo bacterial load as a secondary outcome measure of infection andefficacy of the passive immunization.

Example 3 Kinetics of Infection and Humoral Immunity During theEstablishment of Osteomyelitis

To quantify microbial pathogenesis and host immunity during theestablishment of osteomyelitis, a time course study was performed inwhich mice were given an infected transcortical pin implant in theirtibia, and the bacterial load and the host humoral response wasdetermined over time by nuc/β-actin RTQ-PCR and western blot,respectively (FIGS. 3A-C). The results indicate a clear inversecorrelation between infection and humoral immunity. Consistent withclassical microbial pathogenesis and acquired immunity to extracellularbacteria, these results indicate that the bacteria immediately establishthemselves and enter an exponential growth phase, which is extinguishedby a neutralizing humoral response after 11 days. Based on thecoincidence of the peak bacterial load with the genesis of high affinityIgG antibodies against specific bacterial proteins, it is evident thatthese “immuno-dominant” antigens elicit a functional immune responsethat is both diagnostic and protective against the establishment of OM.

Example 4 Identification and Cloning of the Glucosaminidase Subunit ofS. Aureus Autolysin as 56 kDa Immuno-Dominant Antigen that Elicits aSpecific IgG2b Response During the Establishment of OM

To further characterize the humoral response during the establishment ofOM, the prevalence of Ig isotypes in the serum of mice was determinedover the first two weeks of infection by ELISA (Li et al., “QuantitativeMouse Model of Implant-Associated Osteomyelitis and the Kinetics ofMicrobial Growth, Osteolysis, and Humoral Immunity,” J. Orthop. Res.26(1):96-105 (2008), which is hereby incorporated by reference in itsentirety). The results showed that the mice initiate a classical IgMresponse in the first week that converts to a specific IgG2b response inthe second week, which has recently been shown to have potent opsonicand protective activities against S. aureus antigens (Maira-Litran etal., “Comparative Opsonic and Protective Activities of Staphylococcusaureus Conjugate Vaccines Containing Native or DeacetylatedStaphylococcal Poly-N-acetyl-beta-(1-6)-glucosamine,” Infect. Immun.73(10):6752-62 (2005), which is hereby incorporated by reference in itsentirety).

To elucidate the molecular identity of the immuno-dominant antigensidentified in FIG. 3C, subtractive Western blotting of total S. aureusextract was performed that was separated by 2D-PAGE (FIGS. 4A-C). Thisanalysis revealed a polypeptide that was not detected by the pre-immuneserum, but had strong reactivity with the day 14 post-immune serum. Theprotein was isolated from a preparative Coomassie blue stained gel,digested with trypsin, and analyzed by matrix-assisted laserdesorption/ionization (MALDI), which resolved 70 individual peptidepeaks. The amino acid sequence from every peptide was a 100% match withthe known sequence of the Gmd subunit of S. aureus Alt. Interestingly,others have also recently found Atl to be an immuno-dominant antigen ina rabbit tibia model of MRSA OM (Brady et al., “Identification ofStaphylococcus aureus Proteins Recognized by the Antibody-MediatedImmune Response to a Biofilm Infection,” Infect. Immun. 74(6):3415-26(2006), which is hereby incorporated by reference in its entirety).

To confirm that the spot picked from the 2D-PAGE gel in FIG. 4C was therelevant immuno-dominant antigen, a recombinant 6-His tagged fusionprotein was generated by cloning the 1,465bp coding region of the 53 kDaglucosaminidase subunit of S. aureus autolysin into the XhoI-BamHI siteof the pET-28a(+) expression plasmid (Novagen), which contains the lac Ipromoter for IPTG induction. Following DNA sequencing, the plasmid wasused to transform BL21 E. coli, which were used to make recombinantHis-glucosaminidase (His-Gmd). This recombinant protein was then used toevaluate the reactivity of pre-immune and immune sera. The resultsshowed that the IPTG induced 57 kDa recombinant protein is onlyrecognized by immune serum, thus confirming that Gmd is a S. aureusimmuno-dominant antigen. This experiment was repeated with anti-serafrom mice infected with Xen 29, and confirmed that C57Bl/6 also generateGmd specific antibodies against this bioluminescent strain of S. aureus.

Example 5 In Vivo Bioluminescence Imaging of Lux Transformed S. Aureusas a Longitudinal Outcome Measure of OM and Bacterial Growth

Although the RTQ-PCR method of quantifying OM in mouse model is veryuseful, there are three major limitations to this approach. First, it isnot longitudinal, as analysis requires sacrifice of the mice to harvestthe DNA. Second, it is very labor intense and requires great care duringthe DNA isolation, PCR and data analysis. Third, detection of S. aureusgenomic DNA (nuc genes) cannot distinguish between bacteria that are inan active growth phase vs. dormant bacteria tightly packed in a biofilm.Thus, RTQ-PCR cannot be readily used to assess mAb effect on bacterialgrowth in vivo.

To overcome these shortcomings, the present invention relates to ahighly innovative approach to monitor pathogens in vivo usingbioluminescence imaging (Contag et al., “Photonic Detection of BacterialPathogens in Living Hosts,” Mol. Microbiol. 18(4):593-603 (1995), whichis hereby incorporated by reference in its entirety). More recently, P.R. Contag and colleagues have generated bioluminescent S. aureus (Xen29; ATCC 12600) for this purpose (Francis et al., “MonitoringBioluminescent Staphylococcus aureus Infections in Living Mice Using aNovel luxABCDE Construct,” Infect. Immun. 68(6):3594-600 (2000), whichis hereby incorporated by reference in its entirety). FIGS. 5A-Bdemonstrate how this approach is adapted in the model of OM of thepresent invention. In a time-course studies with Xen29, only backgroundsignal was detected in mice that received a sterile pin or infected micetreated with parenteral gentamycin. In contrast, the BLI of infected,untreated tibiae demonstrated a sharp 4-fold increase from baseline onday 4, which subsequently dropped to background levels by day 11.

Example 6 Recombinant Gmd Vaccine Protects Mice from Implant-AssociatedOM

To assess the potential of an anti-autolysin passive immunization forOM, an initial active recombinant Gmd vaccine study was performed inwhich mice (n=20) were immunized as follows: Group 1 (PBS in adjuvant(negative control)); Group 2 (20 μg S. aureus Xen 29 total proteomeextract emulsified 1:1 with equal volume of adjuvant (positivecontrol)); Group 3 (20 μg His-glucosaminidase in adjuvant). A 150 μlemulsion of each vaccine was injected intramuscularly (i.m.) 28 dayprior to challenge. Booster immunizations (i.m.; 20 μg protein inFreund's incomplete adjuvant) were performed 14 days prior to challenge.

To assess the vaccine efficacy in these mice, an anti-Gmd ELISA wasdeveloped (FIG. 6A) and used to quantify serum antibody titers beforeinitial immunization, before booster immunization, and before thebacterial challenge (FIG. 6B). Remarkably, the results demonstrated thatonly the recombinant vaccine elicited a high titer immune response. Toassess the efficacy of these vaccines, the immunized mice werechallenged with a Xen29 infected transtibial pin as described in thepreceding Example (see FIG. 5A-C), BLI was performed on day 3, and themice were euthanized for nuc RTQ-PCR on day 11. Remarkably, 18 out ofthe 20 mice immunized with S. aureus total proteome died within 48 hr ofthe challenge; thus efficacy data from that group are not available.While only speculative explanations can be provided for this observation(i.e. hyper-immunity to other antigens), the fact that no death occurredin any of the other groups and that the deaths were reproduced in the 4cohorts of 5 mice in Group 2 indicates that the results are real. Forthis reason, this immunization protocol should not be used as a positivecontrol for future studies. It also highlights the safety concerns withactive vaccines, and supports the rationale of a passive immunizationwith purified mAb or binding fragments thereof.

The BLI and nuc RTQ-PCR data from Groups 1 and 3 are presented in FIGS.7A-C. The results clearly demonstrate a significant reduction of BLIdetected in the His-Gmd immunized mice (FIGS. 7A-B), which shows adecrease in planktonic growth of the bacteria. Consistent with thisfinding, it was observed that there was a significant reduction in thenumber of nuc genes at the peak of the bacterial load in this model (day11). Thus, these data demonstrate that the recombinant Gmd vaccine canprotect mice from OM in the model.

Example 7 Generation and Screening of Mouse Anti-Gmd MonoclonalAntibodies

Based on the success of the His-Gmd immunization described in Example 6,this protocol was used to generate mouse anti-Gmd mAb. Standardprocedures were used to generate the mAb. Out of an initial pool ofhybridomas that were prepared, a first subset was selected followingscreened by ELISA for anti-Gmd activity and a second subset possessinghigher affinity were selected following a western dot-blot assay.

Five of the hybridoma cell lines were selected based on their apparenthigh affinity for Gmd (≦10⁻⁹M) and the putative epitope for theseregions being found within the R3 domain of Gmd. Because the R3 domainis not the catalytic domain of the Gmd protein, it was unexpected thatthese monoclonal antibodies would possess as significant anti-Gmdinhibitory activity. The five selected hybridomas were 1C11, 1E12, 2D11,3A8 and 3H6. All secreted mouse IgG1 antibodies.

Example 8 Alteration of In Vitro Growth of Staphylococcus Aureus Xen29by Monoclonal Anti-Glucosaminidase Antibodies

Frozen aliquots of each cell line (1C11, 1E12, 2D11, 3A8 and 3H6) wereobtained directly from the vendor who prepared them at our request(Precision Antibodies, Inc., Columbia, Md.). The frozen cells werethawed, and then washed in Dulbecco's Modified Eagle's Medium (DMEM)supplemented with 50 μg/mL gentamicin and 10% fetal bovine serum (FBS).Harvested culture supernatant was clarified by centrifugation (10 min,1000×g) and frozen.

Thirty mL of culture supernatant from each cell line was thawed at37-45° C. and filtered through a 0.22 μm filter. Each was then purifiedon a 5 mL bed-volume Protein G-agarose column (GE Healthcare HiTrap™Protein G HP, Cat. No. 17-0405-03, Lot #10036021). In the place of thepump for which these columns are designed, fluids were added by means ofluer-lock syringes fitted by adapters to the top of the column. Thecolumn was first washed with PBS to remove the ethanol preservative andthen culture supernatants were added at 5-6 mL/min, followed by sixcolumn volumes of PBS to wash out unbound protein. Adsorbed antibody waseluted with two column volumes of 0.1 M glycine, pH 2.7, into acollection vessel containing 1 mL of 1.0 M Tris, pH 8.0, to neutralizethe eluted product. The column was then washed with four column volumesof PBS in preparation for the next antibody, or with PBS containing0.02% NaN₃ for storage.

The eluted antibodies were concentrated and dialyzed into PBS in Pierce®concentrators (Thermo Scientific 7 mL/20K MWCO, Cat. No. 87750, lot#KH137631A) by successive centrifugations at 3500×g, 40 min, 4° C.Antibody concentration was determined by ELISA using MOPC21 or Phe12.15(both mouse IgG1) as standards. Unlabeled goat antimouse IgG (SouthernBiotechnology Cat. No. 1030-01) was adsorbed onto 96-well NUNC Maxisorpmicrotiter plates at 5 μg/mL in PBS, 100 μL per well, one hour RT orovernight at 4° C. Wells were blocked by the addition of 200 μL of 3%BSA in PBS for one hour RT or overnight at 4° C. Blocked plates werewashed twice with PBS containing 0.05% Tween 20 (PBST) and ready foruse. Samples were prepared as serial dilutions in PBST and 100 μL wasadded to wells designated for standards and samples. The samples wereincubated for one hour, RT, and then washed 4 times with PBST from asquirt bottle. The captured mouse antibody was detected by the additionto each well of 100 μL of HRP-conjugated goat anti-mouse IgG (HRP-GAM;Southern Biotechnology, Cat. No. 1031-05), diluted 1:2000 in PBST, andincubated for one hour, RT. After washing the plates 4 times with PBSTfrom a squirt bottle, the chromogenic HRP substrate ABTS (SouthernBiotechnology, Cat. No. 0401-01) was added, 100 μL per well. Color wasallowed to develop for 5-10 minutes, RT. Antibody concentrations weredetermined by projecting sample color values onto the standard curvesand then correcting for dilution. These concentrations were used fortitration studies to determine their effect on S. aureus growth.

S. aureus Xen29 (Kadurugamuwa et al., “Rapid Direct Method forMonitoring Antibiotics in a Mouse Model of Bacterial Biofilm Infection,”Antimicrob Agents Chemother 47:3130-7 (2003) and Kadurugamuwa et al.,“Noninvasive Optical Imaging Method to Evaluate Postantibiotic Effectson Biofilm Infection In Vivo,” Antimicrob Agents Chemother 48:2283-7(2004), which are hereby incorporated by reference in their entirety)was the only bacterial strain used in these experiments. 1 μL of S.aureus Xen29 was taken from a frozen stock and grown in 10 mL of LBmedium at 37° C. on a rotating platform at 200 rpm for 12 hours tomid-log phase. The bacteria were then diluted in LB medium to 1000cfu/mL and 100 μL of the diluted suspension was added to wells of aflat-bottomed microtiter (with cover) designated for the addition of theantibodies and controls. Each anti-Gmd and control antibody was dilutedinto LB from stocks about 1 mg/mL in PBS and sterilized through a0.2μfilter. 100 μL of each antibody was added to designatedquadruplicate wells. Plates were then incubated at 37° C. and lightscattering was measured at 490 and 670 nm at t=0, 5, 7, 9, 11, 13, 15hours on a microtiter plate reader. A final time point was taken sometime after 24 hours to confirm the measured plateau values.

The five antibodies were purified by affinity chromatography on ProteinG-Agarose, concentrated to 1 mg/mL in PBS and dialyzed to removepreservatives such as NaN₃ and antibiotics that might interfere in theassay. 100 cfu of S. aureus strain Xen29 from a mid-log phase culturewere placed in each microtiter well in LB medium along with 100 μL ofeach antibody or control (also in LB medium) at 50 mg/mL (˜3×10⁻⁷ M).Growth was monitored by measuring light scattering at 490 and 670 nm atintervals over 26 hours.

Apparent reduction in the growth of S. aureus was observed by anti-Gmdmonoclonal antibodies. As depicted in FIG. 8, four of the antibodiesdepressed the growth-related increase in light scattering of Xen29. Thefour antibodies that reduced light scattering all did so to the samedegree while the isotype-matched control (MOPC21) was identical to Xen29grown in the absence of any antibody. The fifth antibody, 3H6, alsodemonstrated similar behavior in a separate experiment.

It was also observed that the alteration of Xen29 growth-related lightscattering is dose-dependent and consistent with high affinityinteraction between monoclonal antibodies and native Gmd. The criticaldecision in addressing dose-dependence of the observed growth alterationof Xen29 was whether the apparent reduction in Xen29 growth was onlypartially revealed because the concentration of antibody was too low orthe maximum effect was already observed and higher concentrations wouldhave no further effect.

Because four of the antibodies had the same magnitude of effect and 50μg/mL (3×10⁻⁷ M) was a very high concentration, the antibody levels weretitrated down from 50 μg/mL in serial 10^(0.5) dilutions. Representativedata is presented in FIG. 9. Monoclonal antibody 1C11 altered the growthof Xen 29 to the same degree at 50, 16 and 5 μg/mL, partially at 1.6(˜1×10⁻⁸ M), and was not different from the irrelevant antibody MOPC21at 0.5 μg/mL (FIG. 10). Essentially identical results were obtained formAbs 2D11, 1E12 and 3A8.

Results for the isotype-matched control antibody MOPC21 are presented inFIG. 10. The growth curves were essentially identical to the no antibodycontrol. The slight elevation of the 50 μg/mL samples was due to theirplacement in outside wells in the microtiter plate (edge effect).

Assuming that the inhibition of Gmd is due to a high degree of antibodybinding, then an estimate assuming that about ten times the K_(d) isrequired places the operational affinity for the Gmd in the vicinity of1 nM.

These data indicate that the four anti-Gmd mAbs inhibit Gmd activity,leading to a change in the in vitro growth pattern of Xen29, but theyhave no effect on the doubling time. The inhibition of Gmd activityleads to failed cytokinesis, i.e., the genome and cell membranes dividenormally, but the cell walls fail to separate leading to large clustersof aggregated cells (Sugai et al., “Identification ofEndo-beta-N-acetylglucosaminidase and N-acetylmuramyl-L-alanine Amidaseas Cluster-dispersing Enzymes in Staphylococcus aureus,” J Bacteriol177:1491-6 (1995), which is hereby incorporated by reference in itsentirety). This change is detectable by light scattering because thereare fewer (albeit larger) scattering centers than when the Xen29 cellsdivide freely.

The antibodies of the present invention have already been shown tosatisfy several key criteria. These are high affinity IgG antibodiesproduced by the parental hybridomas at robust levels (˜50 μg/mL instatic culture). They recognize conserved epitopes and consequently someor most will recognize Gmd from the majority of S. aureus strains. Inaddition, these experiments provide evidence that these monoclonalantibodies recognize native Gmd and not merely the recombinant His-Gmdthat was used as the immunogen, and that they act as Gmd inhibitors. Themonoclonal antibodies all exhibit Gmd-inhibitory activity of betweenabout 70 to about 80 percent. This level of Gmd-inhibitory activity israther surprising given that the five selected antibodies bind toepitopes located within the Gmd R3 domain rather than its catalyticdomain.

Example 9 Monoclonal Antibodies Specific for Glucosaminidase fromStaphylococcus Aureus Use a Diverse Array of V_(H) Genes

To determine that the five monoclonal antibodies under investigationwere unique, the sequences of the V_(H) and V_(L) genes were determinedfor the five candidate hybridomas (1C11, 1E12, 2D11, 3A8 and 3H6)identified in the preceding example.

Because there are considerable medical applications and advantages inthe study of each of a set of monoclonal antibodies specific for the S.aureus glucosaminidase (Gmd), it is beneficial to identify hybridomasexpressing identical antibodies (sibs). Five hybridomas were selectedfor sequence analysis of their Ig heavy and light chain genes.

Frozen aliquots of the five hybridoma cell lines were obtained from thevendor A&G Precision Antibody™ (9130 Red Branch Road, Suite U, Columbia,Md. 21045), who prepared the hybridomas at our direction. Cells werethawed, washed in DMEM with gentamicin and 10% FBS to remove DMSO, andthen cultured in DMEM with gentamicin and 10% FBS. After a few days,cells were harvested by centrifugation and stored at −20° C. as a frozenpellet for subsequent RNA extraction.

RT-PCR amplification of the heavy chain mRNA was successful in the fivehybridomas. Sequence analysis revealed that three different germ-lineV_(H) gene segments were used. Two of the hybridomas (2D11 and 3H6) usedthe same germ-line V_(H) and J segments, but different D-segments andeach displayed only modest sequence diversification from germ-line atthe protein level. The heavy chain from the fifth hybridoma (1C11) didnot initially amplify in RT-PCR even with PCR primers designed toamplify some of the more rarely used V_(H) gene-segments. It can beinferred that it expressed an Ig V_(H) gene distinct from the others.

Total RNA was extracted from freshly growing hybridoma cells and ˜5micrograms of RNA was reverse transcribed using the BioRad iScript kit.Aliquots of cDNA were PCR amplified with consensus primers for the5′-ends of murine variable heavy and light regions paired with constantregion primers. Strong products were obtained for 4/5 V_(H) and 5/5V_(L) genes. The PCR products were gel purified and directly sequenced.All 4 V_(H) products gave clean sequence, but 2/4 light chain productswere mixed. The remaining two gave good sequences. The light chainderived from the cell line that failed to give a heavy chain product wasnot sequenced. The variable regions for those antibodies that failed toamplify in the initial experiments using framework 1 primers weresuccessfully amplified using a different primer set that targets thesecretory leader regions of the variable gene segments. These PCRproducts were also directly sequenced after purification.

Best matches for germ-line V-region genes were determined by using IgBLAST (NCBI). The determined DNA sequence was translated into proteinsequence by using an on-line program available at Expasy website.Sequence alignment of the 2D11 and 3H6 sequences was performed by visualinspection.

As noted above, eventually all of the five V_(H) genes were successfullyamplified and sequenced. Detailed DNA and protein sequence results forthe five hybridomas are presented below.

Hybridoma 2D11 (Closest germ line match: J558.18.108) has the V_(H)nucleotide sequence (SEQ ID NO: 21) as follows:

GAGGTGCAGCTGCAGGAGTCTGGACCTGTGCTGGTGAAGCCTGGGGCTTCAGTGAAGATGTCCTGTAAGGCTTCTGGATACACATTCACTGACTACTATATGAACTGGGTGAAGCAGAGCCATGGAAAGAGCCTTGAGTGGATTGGAGTTATTAATCCTTACAACGGTGATACTACCTACAGCCAGAAGTTCAAGGGCAAGGCCACATTGACTGTTGACAAGTCCTCCAGCACAGCCTACATGGAGCTCAACAGCCTGACATCTGAGGACTCTGCAGTCTATTACTGTGCAAGAAATTACGACGAGTACTTCGATGTCTGGGGCACAGGGACCACGGTCACCGTCTCCTCAGCCAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCNGGTCAAG GGCThe 2D11 V_(L) (Closest germ line match: at4) has the nucleotidesequence (SEQ ID NO: 22) as follows:

GATATTGTGATGACCCAGTCTCCAGCAATCATGTCTGCATCTCCAGGGGAGAAGGTCACCATGACCTGCAGTGCCAGCTCAAGTGTAAGTTACATGTACTGGTACCAGCAGAAGCCAGGATCCTCCCCCAGACTCCTGATTTATGACACATCCAACCTGGCTTCTGGAGTCCCTGTTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTCTCACAATCAGCCGAATGGAGGCTGAGGATGCTGCCACTTATTACTGCCAGCAGTGGAGTAGTTACCCGCTCACGTTCGGTThe amino acid sequence of hybridoma 2D11 V_(H) (SEQ ID NO: 1) is asfollows:

EVQLQESGPVLVKPGASVKMSCKASGYTFTDYYMNWVKQSHGKSLEWIGVINPYNGDTTYSQKFKGKATLTVDKSSSTAYMELNSLTSEDSAVYYCARNYDEYFDVWGTGTTVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCXVKGThe 2D11 V_(L) has the amino acid sequence (SEQ ID NO: 8) as follows:

DIVMTQSPAIMSASPGEKVTMTCSASSSVSYMYWYQQKPGSSPRLLIYDTSNLASGVPVRFSGSGSGTSYSLTISRMEAEDAATYYCQQWSSYPLTFG

Hybridoma 3H6 (Closest germ line match: J558.18.108) has the V_(H)nucleotide sequence (SEQ ID NO: 23) as follows:

GAGGTGCAGCTGCAGGAGTCTGGACCTGTGCTGGTGAAGCCTGGGGCTTCAGTGAAGCTGTCCTGTAAGGCTTCTGGATACACATTCACTGACTACTTTATGAACTGGGTGAAGCAGAGCCATGGAAAGAGCCTTGAGTGGATTGGAGTTATTAATCCTTTCAACGGTGGTAATAGGTACAACCAGAACTTCAAGGGCAAGGCCACATTGACTGTTGACAAGTCCTCCAGCACAGCCTACATGGAGCTCAACAGCCTGACATCTGAGGACTCTGCAGTCTATTACTGTGCAAGAGGGGACTATGACTCCCCCTGGTTTGATTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCAGCCAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTGGTCAAGGGCTATTCCCNGAGCCAGTGThe 3H6 V_(L) (closest germ line match: cp9, JK1) has the nucleotidesequence (SEQ ID NO: 24) as follows:

CAGATGACACAGACTACGTCCTCCCTGTCTGCCTCTCTGGGAGACAGAGTCACCATCAGTTGCAGTGCAAGTCAGGGCATTAGCAATTATTTAAACTGGTATCAGCAGAAACCAGATGGAACTGTTAAACTCCTGATCTATTACACATCAAGTTTACACTCAGGAGTCCCATCAAGGTTCAGTGGCGGTGGGTCTGGGACAGATTATTCTCTCTCCATCAGCAACCTGGAACCTGAAGATATTGCCACTTACTATTGTCAGCAGTATAGTAAGCTTCCTTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAAThe amino acid sequence of hybridoma 3H6 V_(H) (SEQ ID NO: 2) is asfollows:

EVQLQESGPVLVKPGASVKLSCKASGYTFTDYFMNWVKQSHGKSLEWIGVINPFNGGNRYNQNFKGKATLTVDKSSSTAYMELNSLTSEDSAVYYCARGDYDSPWFDYWGQGTLVTVSAAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYSXSQThe amino acid sequence of hybridoma 3H6 V_(L) (SEQ ID NO: 9) is asfollows:

QMTQTTSSLSASLGDRVTISCSASQGISNYLNWYQQKPDGTVKLLIYYTSSLHSGVPSRFSGGGSGTDYSLSISNLEPEDIATYYCQQYSKLPWTF GGGTKLEIK

Hybridoma 1E12 (Closest germ line match: 7183.46 VH7) has the V_(H)nucleotide sequence (SEQ ID NO: 25) as follows:

GAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTCGTGAAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTACCTATGTCATGTCTTGGGTTCGCCAGACTCCGGAAAAGAGGCTGGAGTGGGTCGCAACCATTAGTGATGGTGGTGGTCATACTTACTATCTAGACAATGTAAAGGGCCGATTCACCATCTCCAGAGACAATGCCAAGAACAACCTGTACCTGCACATGAGCCATCTGAAGTCTGAGGACACAGCCATGTATTACTGTGCAAGAGCTTACTACGGTAGTAGTTACGACGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAGCCAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTGGTCAAGGGCThe 1E12 V_(L) (Closest germ line match: ai4) has the nucleotidesequence (SEQ ID NO: 26) as follows:

GATATTGTGATCACCCAGTCTCCAGCAATCATGTCTGCATCTCTAGGGGAACGGGTCACCATGACCTGCACTGCCAGCTCAAGTGTAAGTTCCAGTTACTTACACTGGTACCAGCAGAAGCCAGGATCCTCCCCCAAACTNTGGATTTATAGCACATCCAACCTGGCTTCTGGAGTCCCAGCTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTCTCACAATCAGCAGCATGGAGGCTGAAGATGCTGCCACTTATTACTGCCACCAGTATCATCGTTCCCCA TGGACGTTCGGTGGAGGCACCThe amino acid sequence of hybridoma 1E12 V_(H) (SEQ ID NO: 3) is asfollows:

EVQLQESGGGFVKPGGSLKLSCAASGFTFSTYVMSWVRQTPEKRLEWVATISDGGGHTYYLDNVKGRFTISRDNAKNNLYLHMSHLKSEDTAMYYCARAYYGSSYDAMDYWGQGTSVTVSSAKTTPPSVYPLAPGSAAQTNSMV TLGCLVKGThe 1E12 V_(L) has the amino acid sequence (SEQ ID NO: 10) as follows:

DIVITQSPAIMSASLGERVTMTCTASSSVSSSYLHWYQQKPGSSPKXWIYSTSNLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCHQYHRSP WTFGGGT

Hybridoma 3A8 (Closest germ line match: VHJ606.4.8.2) has the V_(H)nucleotide sequence (SEQ ID NO: 27) as follows:

GAGGTGCAGCTGCAGGAGTCTGGAGGAGGCTTGGTGCAACCTGGAGGATCCATGAAACTCTCTTGTGCTGCCTCTGGATTCACTTTTAGTGACGCCTGGATGGACTGGGTCCGCCAGTCTCCAGAGAAGGGGCTTGAGTGGGTTGCTGAAATTAAAGACAAAACTAATAATCATGCAACATACTATGCTGAGTCTGTGAAAGGGAGGTTCACCATCTCAAGAGATGTTTCCAAAAGTCGTGTCTTCCTGCAAATGAACAGCTTAAGACCTGAAGACACTGGCATTTATTACTGTACGTCTGGGCCATATTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCAGCCAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTGGTCAAGGGCTATTTCCCTGAGThe 3A8 V_(L) (closest germ line match: KV 19-25, JK2) has thenucleotide sequence (SEQ ID NO: 28) as follows:

GACATTGTGATGACCCAGTCTCACAAATTCATGTCCACATCAGTAGGAGACAGGGTCAGCATCACCTGCAAGGCCAGTCAGGACGTGAGTACTGCTGTAGCCTGGTATCAACAAAAACCAGGGCAATCTCCTAAACTACTGATTTACTGGACATCCACCCGGCACACTGGAGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTTACTCTCACCATCAGCAGTGTGCAGGCTAAAGACCTGGCACTTTATTACTGTCAGCAACATTATACCACTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAAThe amino acid sequence of hybridoma 3A8 V_(H) (SEQ ID NO: 4) is asfollows:

EVQLQESGGGLVQPGGSMKLSCAASGFTFSDAWMDWVRQSPEKGLEWVAEIKDKTNNHATYYAESVKGRFTISRDVSKSRVFLQMNSLRPEDTGIYYCTSGPYFDYWGQGTTLTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGC LVKGYFPEThe amino acid sequence of hybridoma 3A8 V_(L) (SEQ ID NO: 11) is asfollows:

DIVMTQSHKFMSTSVGDRVSITCKASQDVSTAVAWYQQKPGQSPKLLIYWTSTRHTGVPDRFTGSGSGTDFTLTISSVQAKDLALYYCQQHYTTPY TFGGGTKLEIK

Hybridoma 1C11 (closest germline match: VH 9-15, DST4-057B1-6, JH3) hasthe V_(H) nucleotide sequence (SEQ ID NO: 29) as follows:

CAGATCCAGTTGGTACAGTCTGGACCTGAGCTGAAGAAGCCTGGAGAGACAGTCAAGATCTCCTGCAAGGCTTCTGGGTATACCTTCACAACGTATGGAATGAGCTGGGTGAATCAGGCTCCAGGAAAGGGTTTAAAGTGGATGGGCTGGATAAACACCTACTCTGGAGTGCCAACATATGCTGATGACTTCAAGGGACGGTTTGTCTTCTCTTTGGAAACCTCTGCCAGCACTGCCTATTTGCAGATCAACAACCTCAAAAATGAGGACACGGCTACATATTTCTGTGCAAGAGAGGAGTACAGCTCAGGCTACGCGGCCTGGTTTCCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCAThe 1C11 V_(L) (closest germ line match: VK23-43, JK5) has thenucleotide sequence (SEQ ID NO: 30) as follows:

GATATTGTGCTAACTCAGTCTCCAGCCACCCTGTCTGTGACTCCAGGAGATAGCGTCAGTCTTTCCTGCAGGGCCAGCCAAAGTATTAGCAACAACCTACACTGGTATCAACAAAAATCACATGAGTCTCCAAGGCTTCTCATCGAATATGCTTCCCGGTCCATCTCTGGGATCCCCTCTAGGTTCAGTGGCGGTGGATCAGGGACAGATTTCACTCTCAGTATCAACAGTGTGGAGTCTGAAGATTTTGGATTGTATTTCTGTCAACAGAGTAACAGCTGGCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAAThe amino acid sequence of hybridoma 1C11 V_(H) (SEQ ID NO: 5) is asfollows:

QIQLVQSGPELKKPGETVKISCKASGYTFTTYGMSWVNQAPGKGLKWMGWINTYSGVPTYADDFKGRFVFSLETSASTAYLQINNLKNEDTATYFCAREEYSSGYAAWFPYWGQGTLVTVSAThe amino acid sequence of hybridoma 1C11 V_(L) (SEQ ID NO: 12) is asfollows:

DIVLTQSPATLSVTPGDSVSLSCRASQSISNNLHWYQQKSHESPRLLIEYASRSISGIPSRFSGGGSGTDFTLSINSVESEDFGLYFCQQSNSWPL TFGAGTKLELK

From the obtained sequences the closest fit for germ line V_(H) andV_(L) gene segments was determined as shown in Table 1.

TABLE 1 Germline Matches for Sequenced Hybridoma Cell Lines HybridomaGerm Line V_(H) Germ Line V_(L) 1C11 VH 9-15, DST4-C57B1-6, JH3 VK23-43,JK5 1E12 7183.46 VH7 ai4 2D11 J558.18.108 at4 3A8 VHJ606.48.2 KV 19-25,JK2 3H6 J558.18.108 cp9, JK1

Each of the five hybridomas expressed a unique V_(H) gene. FIG. 11 showsthe alignment of the four initially sequenced V_(H) genes, and FIG. 13shows the alignment of all five sequenced V_(H) genes. FIG. 15 shows thealignment of the sequenced V_(H) genes for 1C11 and 3A8 only.

FIG. 12 shows the alignment of the two initially obtained V_(L) genes.FIG. 14A shows the alignment of all five sequenced V₁ genes, whereasFIG. 14B all five sequenced V₁ genes except of that for 1C11.

The fact that one hybridoma (1C11) did not yield a PCR product with theinitial primers that amplify the most common V_(H) genes suggests thatit uses a fourth V_(H) gene segment not identical to any of the othersidentified here. Two of the hybridomas used the same V_(H) germ linegene, but are not identical. Hybridomas 2D11 and 3H6 both used the germline V_(H) gene segment J558.18.108. Their sequences are compared withthe germ line gene in FIG. 11. Inspection of the sequence reveals thatthere is only one difference in CDR1, four in CDR2 and five in CDR3(including gaps).

The five hybridomas are not sibs and among them at least four V_(H) germline genes were utilized.

Example 10 Inhibition of S. aureus Gmd Enzymatic Activity by mAbs 1C11,2D11, 3H6, 1E12, and 3A8

The method of measurement of Gmd enzymatic activity is essentially themethod of Mani et al. (Mani et al., “Isolation and Characterization ofAutolysis-Defective Mutants of Staphylococcus aureus Created byTn917-lacZ Mutagenesis” J. Bacteriol. 175(5): 1493-1499 (1993)).Lyophilized and resuspended Micrococcus lysodeikticus were degraded bythe action of the Gmd resulting in a reduction in light scattering at490 nm. 100 μL of sample containing Gmd diluted in phosphate-bufferedsaline with 0.05% Tween 20 (PBST) was added to the wells of a 96-wellmicrotiter plate. 100 μL of a 0.15% (w/v) suspension of MIcrococcuslysodeikticus was added to each well and the light scattering wasmeasured immediately to establish the initial A₄₉₀, typically about 0.8.The plate was then incubated at 37° C. and light scattering wasre-measured at 30 and 60 minutes. Reduction in A₄₉₀ at 60 minutes wastaken as the measure of Gmd activity. The modest background rate (noGmd) was subtracted. This method does not distinguish Gmd activity fromlysozyme activity.

To measure inhibition of Gmd enzymatic activity the Gmd was pre-titeredto determine the concentration that will yield about a 50% reduction inA₄₉₀ in 60 minutes. Then, 50 μL of antibody diluted in PBST was added toeach well of a 96-well microtiter plate followed by 50 μL ofappropriately diluted Gmd, and the mixtures were allowed to incubate for5 or more minutes, and finally 100 μL of 0.15% MIcrococcus lysodeikticuswas added and the initial A₄₉₀ was measured. The plate was thenincubated at 37° C. and the A₄₉₀ was measured at 30 and 60 minutes.Percent inhibition was calculated as 100*(1−(Δ₆₀A₄₉₀ inhibitor/Δ₆₀A₄₉₀no inhibitor control)).

In FIG. 18, serial dilutions of each of the five antibodies plus theisotype-matched negative control MOPC21 were assessed for their abilityto inhibit the catalytic activity of recombinant S. aureus His-Gmd (Gmd)or hen egg lysozyme (HEL). Each of the five anti-Gmd monoclonalantibodies inhibited His-Gmd activity by 75-80%, while displaying noinhibitory activity with HEL. MOPC21 had no inhibitory activity witheither enzyme. The degree of inhibition of His-Gmd has not been observedto exceed 80% by these five antibodies. High, though partial, inhibitionis one of the characteristics of this group of antibodies.

The ability of the five antibodies to inhibit the native Gmd enzymesecreted by S. aureus strain UAMS-1 is depicted in FIG. 19. As with therecombinant Gmd, the native Gmd was inhibited about 80% by eachantibody. By the measure of enzyme inhibition, the antibodies reactsimilarly with both the native and recombinant Gmds.

An SEM analysis of anti-Gmd activity for several monoclonal antibodiesis shown in FIGS. 20C-D. Xen29 S. aureus was grown for 12 hours inLuria-Bertani broth to achieve a mid-log growth suspension, and then10,000 CFU were incubated with: (FIGS. 20A-B) no antibody, (FIG. 20C) 50μg/ml 1E12, or (FIG. 20D) 50 μg/ml 1C11 for 1 hour. Samples were thenplated onto sterile silicon chips, fixed, dehydrated, and coated withgold for visualization by scanning electron microscopy. Micrographs(FIGS. 20C-D) illustrate the effect of anti-Gmd antibody, promotingformation of large clusters and cell-independent lysis (arrows) ofapproximately 20% of the cells.

Example 11 Passive Vaccine Containing Anti-Gmd mAbs InhibitsStaphylococcus Aureus in Vivo Following Orthopedic Implant in Mouse OMModel

The OM model with trans-tibial pin (see Examples 1 and 6) was used toassess the ability of candidate mAbs 1C11 and 3A8 to inhibit S. aureusgrowth in vivo. Briefly, five week old female BALB/cJ mice received anintraperitoneal injection of saline (n=10) or 1 mg of purified 3A8anti-Gmd antibody (n=5) in 0.25 ml saline or 31C11 A8 anti-Gmd antibody(n=5) in 0.25 ml saline 3 days prior to surgery. At surgery, the micereceived a transtibial implant containing 500,000 CFU of Xen29 S.aureus. The mice were imaged to assess bioluminescence on days 0, 3, 5,7, 10 or 11, and 14, and images with the BLI heat map from arepresentative animal in each group are shown in FIGS. 21A and 22A. Ofnote is the absence of a BLI signal in the anti-Gmd 3A8 animal until day11 and 1C11 animal until day 5, presumably when the antibody titerdecreased below the effective concentration. The BLI values on day 3 foreach mouse in the study are shown with the mean for each group (FIG.21B, p=0.02; FIG. 22B). For 1C11, it is interesting to note that thistherapy cured 50% of the animals at day 3. X-rays from a representativeanimal in each group obtained on day 14 is shown to illustrate theosteolytic lesion (arrow) in the placebo mouse, which was not present inthe anti-Gmd treated animals (FIGS. 21C, 22C).

Example 12 Generation of Humanized Antibody

The variable regions of the light and heavy chains of the 1C11 antibodywere re-amplified from the purified hybridoma PCR product described inExample 9 using primers to permit cloning into the human antibodyexpression vectors described by Tiller et al. (“Efficient Generation ofMonoclonal Antibodies from Single Human B Cells by Single Cell RT-PCRand Expression Vector Cloning,” J. Immunol. Methods 329(1-2):112-24(2008), which is hereby incorporated by reference in its entirety).Plasmids containing the 1C11 light and heavy chain variable regions andhuman kappa and IgG1 constant regions were prepared and co-transfectedinto HEK293 cells. After 3 days, the medium was removed from the cellsand assayed for the presence of human IgG and for binding to immobilizedGmd protein by ELISA. Bound antibody was detected using a goatanti-Human IgG antibody coupled to horseradish peroxidase and 3,3′,5,5′tetramethylbenzidene substrate.

To establish that the human:mouse chimeric 1C11 (h1C11) reacted with Gmdas well as the parental mouse 1C11, each was tested for its ability toinhibit the enzymatic activity of His-Gmd. Both h1C11 and mouse 1C11displayed nearly identical inhibitory activity (FIG. 23), therebydemonstrating that the chimeric IgG molecule retained the bindingactivity of the parent.

A similar procedure will be performed using the human CDR1 and CDR2homologs of 1C11 identified in FIGS. 17A-B, and a CDR3 region from oneor more candidate D regions including, without limitation, IGHD5-5, 18,and 12*01.

The humanized 1C11 antibody and antibody comprising the human CDR1 andCDR2 homologs of 1C11 can be utilized in a phase I clinical trial inelderly patients (>65 yrs) undergoing primary total joint replacement.

Although preferred embodiments have been depicted and described indetail herein, it will be apparent to those skilled in the relevant artthat various modifications, additions, substitutions, and the like canbe made without departing from the spirit of the invention and these aretherefore considered to be within the scope of the invention as definedin the claims which follow.

1. A monoclonal antibody or binding portion thereof that bindsspecifically to a Staphylococcus aureus glucosaminidase and inhibits invivo growth of S. aureus. 2-3. (canceled)
 4. The monoclonal antibodyaccording to claim 1, wherein the antibody or binding portion binds toan epitope wholly or partly within the Staphylococcus aureusglucosaminidase R3 domain.
 5. The monoclonal antibody according to claim1, wherein the monoclonal antibody binds to a glucosaminidasepolypeptide fragment that spans amino acids 776-873, 776 to 931,776-1016 or 842-948 of autolysin, as depicted in SEQ ID NO:
 36. 6. Themonoclonal antibody according to claim 1, wherein the antibody comprisesa V_(H) domain comprising one of the following amino acid sequences:(SEQ ID NO: 1) EVQLQESGPVLVKPGASVKMSCKASGYTFTDYYMNWVKQSHGKSLEWIGVINPYNGDTTYSQKFKGKATLTVDKSSSTAYMELNSLTSEDSAVYYCARNYDEYFDVWGTGTTVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGC XVKG; or (SEQ ID NO: 2)EVQLQESGPVLVKPGASVKLSCKASGYTFTDYFMNWVKQSHGKSLEWIGVINPFNGGNRYNQNFKGKATLTVDKSSSTAYMELNSLTSEDSAVYYCARGDYDSPWFDYWGQGTLVTVSAAKTTPPSVYPLAPGSAAQTNSMVTL GCLVKGYSXSQ,where X is any amino acid; or (SEQ ID NO: 3)EVQLQESGGGFVKPGGSLKLSCAASGFTFSTYVMSWVRQTPEKRLEWVATISDGGGHTYYLDNVKGRFTISRDNAKNNLYLHMSHLKSEDTAMYYCARAYYGSSYDAMDYWGQGTSVTVSSAKTTPPSVYPLAPGSAAQTNSMV TLGCLVKG; or(SEQ ID NO: 4) EVQLQESGGGLVQPGGSMKLSCAASGFTFSDAWMDWVRQSPEKGLEWVAEIKDKTNNHATYYAESVKGRFTISRDVSKSRVFLQMNSLRPEDTGIYYCTSGPYFDYWGQGTTLTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGC LVKGYFPE; or(SEQ ID NO: 5) QIQLVQSGPELKKPGETVKISCKASGYTFTTYGMSWVNQAPGKGLKWMGWINTYSGVPTYADDFKGRFVFSLETSASTAYLQINNLKNEDTATYFCAREEYSSGYAAWFPYWGQGTLVTVSA, where X is any amino acid; or (SEQ ID NO: 6)XXQLXXSGXXXXXPGXXXKXSCXASGXTFXXXXMXWVXQXXXKXLXWXXXIXXXXXXXXXXYXXXXKGXXXXXXXXXXXXXXXXXXXXLXXEDXXXYXCXXXXYXXXXXXXXXXWGXGTXXTVSXXXXXXXXXXXXXXXXXXXX XXXXXXXXX,where X is any amino acid or deletion thereof; or (SEQ ID NO: 31)EVQLQESGXXXVXPGXSXKXSCXASGXTFXXXXMXWVXQXXXKXLEWXXXIXXXXXXXXXXYXXXXKGXXTXXXDXXXXXXXXXXXXXLXXEDXXXYYCXXXXYXXXXXXXDXWGXGTXXTVSXAKTTPPSVYPLAPGSAAQTN SMVTLGCXVKGXXXXX,where X is any amino acid or deletion thereof; or (SEQ ID NO: 7)XXQLXXSGXXLXXPGXXXKXSCXASGXTFXXXXMXWVXQXPXKGLXWXXXIXXXTXXXXYAXXXKGRFXXSXXXSXSXXXLQXNXLXXEDTXXYXCXXXXXXSGXXXXFXYWGQGTXXTVSXXXXXXXXXXXXXXXXXXXXXXX XXXXXXXXXXXXXX,where X is any amino acid or deletion thereof; or (SEQ ID NO: 34)QXQLVQSGXEXKXPGXXVKXSCKASGYXFTTYGMXWVXQAPGXGLXWMGWXNTYXGXPTYAXXFXGRFVFSXXTSASTAYLQIXXLKXEDXAXYXCARXXXXXXXXXXXXYWGQGTLVTVSA,where X is any amino acid or deletion thereof;

or wherein the antibody comprises a V_(L) domain comprising one of thefollowing amino acid sequences: (SEQ ID NO: 8)DIVMTQSPAIMSASPGEKVTMTCSASSSVSYMYWYQQKPGSSPRLLIYDTSNLASGVPVRFSGSGSGTSYSLTISRMEAEDAATYYCQQWSSYPLT FG; or (SEQ ID NO: 9)QMTQTTSSLSASLGDRVTISCSASQGISNYLNWYQQKPDGTVKLLIYYTSSLHSGVPSRFSGGGSGTDYSLSISNLEPEDIATYYCQQYSKLPWTF GGGTKLEIK; or(SEQ ID NO: 10) DIVITQSPAIMSASLGERVTMTCTASSSVSSSYLHWYQQKPGSSPKXWIYSTSNLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCHQYHRSP WTFGGGT,where X is any amino acid; or (SEQ ID NO: 11)DIVMTQSHKFMSTSVGDRVSITCKASQDVSTAVAWYQQKPGQSPKLLIYWTSTRHTGVPDRFTGSGSGTDFTLTISSVQAKDLALYYCQQHYTTPY TFGGGTKLEIK; or(SEQ ID NO: 12) DIVLTQSPATLSVTPGDSVSLSCRASQSISNNLHWYQQKSHESPRLLIEYASRSISGIPSRFSGGGSGTDFTLSINSVESEDFGLYFCQQSNSWPL TFGAGTKLELK; or(SEQ ID NO: 13) DIXXTQXXXXXSXXXGXXVXXXCXASXXXSXXXXXWYQQKXXXXXXXXIXXXSXXXXGXPXRFXGXGSGTXXXLXIXXXXXXDXXXYXCXQXXXXP XTFGXGTXXXXX,where X is any amino acid or deletion thereof; or (SEQ ID NO: 14)DIVXTQSXXXXSXXXGDXVROCCXASQXXSXXXXWYQQKXXXSPXLLIXXXSXXXXGXPXRFXGXGSGTDFTLXIXSVXXXDXXLYXCQQXXXXPX TFGXGTKLEXK,where X is any amino acid or deletion thereof; or (SEQ ID NO: 32)DIVXTQSPAIMSASXGEXVTMTCXASSSVSXXYXXWYQQKPGSSPXXXIYXTSNLASGVPXRFSGSGSGTSYSLTISXMEAEDAATYYCXQXXXXP XTFGXXX,where X is any amino acid or deletion thereof; or (SEQ ID NO: 33)DIXXTQXXXXXSXSXSXXSXXXSXSSXXXSXXXXXWYQQKPXXXXXXXIYXTSXXXXGVPPXRFXGXGSGTXXXLXISXXXXXDXAXYYCXQXXXX PXTFGGGTXXXXX,where X is any amino acid or deletion thereof; or (SEQ ID NO: 35)XIVLTQSPXXXSVTPXXXVXXXCRASQSIXXXLHWYQQXXXXSPXLLIXYASXSXSGXPSRFSGXGSGTDFTLXINSXEXEDXXXYXCXQSXSXPL TFGXGTKXEXK,where X is any amino acid or deletion thereof.


7. The monoclonal antibody according to claim 1, wherein the monoclonalantibody is produced by a hybridoma cell line designated 1C11, 1E12,2D11, 3A8, or 3H6.
 8. The monoclonal antibody according to claim 1,wherein the monoclonal antibody promotes cell-independent lysis of S.aureus.
 9. The monoclonal antibody according to claim 1, wherein themonoclonal antibody inhibits activity of Gmd by about 70 to about 80%.10. The monoclonal antibody according to claim 1, wherein the in vivogrowth inhibition is measured using an animal model implanted with atranstibial implant infected with 500,000 CFU of a bioluminescent S.aureus.
 11. The monoclonal antibody according to claim 1, wherein themonoclonal antibody is humanized.
 12. (canceled)
 13. The monoclonalantibody binding portion according to claim 1, wherein the bindingportion comprises a Fab fragment, Fv fragment, single-chain antibody, aV_(H) domain, or a V_(L) domain.
 14. A cell line that expresses amonoclonal antibody according to claim 1 or a binding portion thereof.15. The cell line according to claim 14, wherein the cell line ishybridoma 1C11, 1E12, 2D11, 3A8, or 3H6.
 16. A pharmaceuticalcomposition comprising a carrier and one or more monoclonal antibodiesaccording to claim 1 or one or more binding portions of said monoclonalantibodies.
 17. (canceled)
 18. The pharmaceutical composition accordingto claim 16 further comprising an antibiotic agent or immunotherapeuticagent.
 19. The pharmaceutical composition according to claim 18, whereinthe antibiotic agent is selected from the group consisting ofvancomycin, tobramycin, cefazolin, erythromycin, clindamycin, rifampin,gentamycin, fusidic acid, minocycline, co-trimoxazole, clindamycin,linezolid, quinupristin-dalfopristin, daptomycin, tigecycline,dalbavancin, telavancin, oritavancin, ceftobiprole, ceftaroline,iclaprim, and the carbapenem CS-023/RO-4908463.
 20. The pharmaceuticalcomposition according to claim 18, wherein the immunotherapeutic agentis tefibazumab, BSYX-A110, or Aurexis™.
 21. A method of introducing anorthopedic implant into a patient comprising: administering to a patientin need of an orthopedic implant an effective amount of a monoclonalantibody according to claim 1 or a binding portion thereof; andintroducing the orthopedic implant into the patient.
 22. The methodaccording to claim 21, further comprising repeating said administeringprior to said introducing.
 23. The method according to claim 21, furthercomprising repeating said administering after said introducing.
 24. Themethod according to claim 21, wherein said administering is carried outsystemically.
 25. The method according to claim 21, wherein saidadministering is carried out directly to a site of implantation.
 26. Themethod according to claim 21, wherein the orthopedic implant is a jointprosthesis, graft or synthetic implant. 27-28. (canceled)
 29. The methodaccording to claim 21, further comprising administering a secondtherapeutic agent to the patient, wherein the second therapeutic agentis an antibiotic agent or immunotherapeutic agent.
 30. The methodaccording to claim 29, wherein the antibiotic agent is selected from thegroup consisting of vancomycin, tobramycin, cefazolin, erythromycin,clindamycin, rifampin, gentamycin, fusidic acid, minocycline,co-trimoxazole, clindamycin, linezolid, quinupristin-dalfopristin,daptomycin, tigecycline, dalbavancin, telavancin, oritavancin,ceftobiprole, ceftaroline, iclaprim, and the carbapenemCS-023/RO-4908463.
 31. The method according to claim 29, wherein theimmunotherapeutic agent is tefibazumab, BSYX-A110, or Aurexis™.
 32. Themethod according to claim 21, wherein the orthopedic implant is a jointprosthesis for revision total joint replacement, said method furthercomprising: removing an infected joint prosthesis from the patient andtreating the patient for the infection prior to said introducing theorthopedic implant into the patient.
 33. The method according to claim32, wherein said treating comprises administering an effective amount ofan antibiotic agent, said monoclonal antibody or binding portionthereof, or a combination thereof.
 34. The method according to claim 32,wherein said method is effective to prevent infection or reinfectionduring the revision total joint replacement.
 35. A method of treating S.aureus infection comprising: administering to a patient having a S.aureus infection an effective amount of a monoclonal antibody accordingto claim 1 or a binding portion thereof, wherein said administering iseffective to treat the infection. 36-42. (canceled)
 43. A method oftreating osteomyelitis comprising: administering to a patient having aS. aureus bone or joint infection an effective amount of a monoclonalantibody according to claim 1 or a binding portion thereof, wherein saidadministering is effective to treat the osteomyelitis. 44-49. (canceled)50. The method according to claim 21, wherein the patient is older than50 years of age or immunocompromised.